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Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in Escherichia coli was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli . The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.

Bovine viral diarrhea virus (BVDV) is a primary pathogen responsible for bovine enteric, respiratory and reproductive failure. A genetic region is encoding the p80 (NS3) of BVDV as the most conserved protein among Pestiviruses. BVDV infection in cattle induces NS3 specific lymphocyte proliferation and humoral responses. To generate a DNA vaccine against BVDV, the gene for BVDV-NADL NS3 was cloned into an eukaryotic expression vector of Semiliki Forest virus (pSFV-1). Quadriceps muscles of BALB/c mice were injected with recombinant DNA generated statistically significant cytotoxic T-lymphocyte activity (CTL) and cell mediated immune (CMI) responses against cytopathic and noncytopathic BVDV. Whereas, the BVDV-NS3 did not generate neutralizing antibodies against BVDVin mice. pSFV-1-NS3 DNA was subjected to in vitro transcription into mRNA. The mRNA was transfected into baby hamster kidney cells (BHK-21) and Madin–Darby bovine kidney cells (MDBK). The recombinant cells were used in the detection of DNA antigen responses by immunological assays. This report establishes the ability of BVDV-NS3 DNA inoculation to induce a strong cellular immune responses in mice.

Baculovirus (Autographa californica nuclear polyhedrosis)-expressed bovine herpesvirus-1 (BHV-1) glycoproteins B (gB), gC, and gD were developed and characterized. The recombinant proteins retained their antigenic properties as determined by immunoblotting against monoclonal antibodies. The proteins were examined as antigens for detection of BHV-1 infection and for the analysis of antibody responses to the individual viral proteins. A total of 115 bovine serum samples were tested for their reactivity with individual recombinant proteins from baculovirus-infected Spodoptera frugiperda (SF-9) cell lysates by enzyme-linked immunosorbent assay (ELISA), western blotting, and dot blotting assays. These serum samples were previously tested for BHV-1-specific antibodies by virus neutralization (VN) at the veterinary diagnostic laboratory. All 90 serum samples tested positive with VN were positive by ELISA against gC and gD, separately. However, reactivities of sera against gB were generally low and inconsistent. On the other hand, out of 25 sera that were negative with VN, 22 sera were consistent and gave negative results against gC or gD by ELISA, whereas reactivities with gB were inconsistent. Similar results were obtained when the sera were tested by western blotting and dot blotting. The positive sera consistently reacted strongly against gC and to a lesser extent gD. These results suggest that baculovirus expressed gC from infected cell lysate can be used as a potential diagnostic antigen for detection of anti-gC-specific antibody responses in BHV-1 infected cattle.

The effects of cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhea virus (BVDV) on the cellular metabolic activity and activation status of bovine peripheral blood mononuclear cells (PBMC) were investigated. Cellular DNA and protein synthesis was determined by [3H]thymidine and [3H]valine incorporation, respectively, in phytohemagglutinin (PHA)-stimulated PBMC. All cp strains and most ncp BVDV strains significantly inhibited DNA synthesis in PHA-stimulated PBMC; however, only cp BVDV strains inhibited protein synthesis. A plaque assay and immunofluorescence test confirmed productive BVDV infection of PBMC. In addition, viral RNA synthesis was demonstrated in BVDV-infected PBMC by RT-PCR. The interleukin-2 receptor (IL-2R) was used as a marker for the activation status of BVDV-infected PBMC. The expression of IL-2R was preserved in virus-infected cells, even though DNA and protein synthesis was suppressed. These findings suggest a novel mechanism of virus-induced immune suppression in which BVDV inhibits basic metabolic activities of bovine PBMC. The activation signals, however, are maintained.

A western blot assay using purified recombinant bovine immunodeficiency virus gag protein has been developed for detection of bovine immunodeficiency virus antibodies in bovine serum samples. The test was standardized with known bovine immunodeficiency virus positive and negative bovine serum samples and the monoclonal antibody to gag protein. Both naturally and experimentally infected cattle sera demonstrated positive test results. The result of western blot assay was compared with polymerase chain reaction test results in 134 blood samples collected from Kansas. Twenty-six samples tested positive for bovine immunodeficiency virus DNA with polymerase chain reaction (18.7%) and 25 were positive for the antibody to gag protein by western blot analysis (17.9%). Of 26 cattle testing positive using the polymerase chain reaction assay, 24 were antibody-positive by western blot assay, thus establishing a strong correlation between the two tests. The sensitivity and specificity of western blot relative to polymerase chain reaction are 0.92 and 0.99, respectively. The western blot assay proved to be a specific and sensitive test.

In a previous communication, we reported a 50-kDa cell surface protein from Madin-Darby bovine kidney (MDBK) cells as a putative receptor for bovine viral diarrhea virus (BVDV). The present study delineates further characterization of the receptor protein. Protease treatment of cultured MDBK cells adversely affected the receptor, thus abolishing the binding of anti-D89 (BVDV anti-idiotypes) to the cells. However, pretreatment of the cells with either phospholipases or glycosidases did not significantly change the anti-D89 binding to the cells. Additionally, pretreatment of cell monolayers with proteases decreased BVDV attachment and replication in the cells. These results suggested that the receptor for BVDV is a protein in nature, and glycosylation and phosphorylation may not play a direct role in BVDV attachment to cells. The BVDV receptor gradually regenerated on the cell surface after the protease-treated cells were cultured in normal growth medium. Regeneration of the BVDV receptor to a normal level took about 4 h as indicated by flow cytometric analysis and this process was inhibited in the presence of cycloheximide, a protein synthesis inhibitor. The 50-kDa receptor protein purified by electro-elution inhibited BVDV infection in a plaque reduction assay. It also inhibited anti-D89 binding to cells as analyzed by flow cytometry. These data demonstrated the nature of the 50-kDa protein as a specific receptor for BVDV.

Anti-idiotypic antibodies (anti-ids) have been used successfully in studies on bovine viral diarrhea virus (BVDV) receptor(s) in our laboratory. The anti-ids specifically bound to cultured cells and identified a 50 kDa cellular membrane protein, which is thought to be a specific receptor for BVDV. In this study, flow cytometric analyses demonstrated that the anti-ids also specifically bound to different cell types, namely MDBK, EBK, BT, PK15, MA1O4, and Vero. Experiments on virus attachment and replication showed that BVDV adsorbed to all cells and replicated in them except monkey kidney cells MA 104 and Vero (non-permissive). Results from plaque reduction assays indicated that cellular membrane proteins from all cell lines competitively inhibited BVDV attachment to cultured MDBK cells, suggesting the presence of BVDV receptor on all cells. Immunoblotting of cell membrane proteins with the anti-ids revealed a 50 kDa protein in both permissive and nonpermissive cells. Subcloned or synchronized MDBK cells demonstrated no significant difference of binding with anti-ids as compared to normal cultured cells.

Overlapping fragments of the bovine herpesvirus-1 (BHV-1) glycoprotein (gD) ORF were expressed as trpE-gD fusionproteins in Escherichia coli to map linear neutralizing epitopes defined by BHV-1-specific MAbs. The MAbs 3402 and R54 reacted with the expressed fragments on Western blots that located the epitopes between the amino acids 52–126 and 165–216, respectively, of gD. Bovine covalescent sera with high neutralizing antibody titers against BHV-1 reacted with these bacterially expressed proteins containing both of the epitopes. Alignment of these sequences from BHV-1 with the corresponding region of the BHV-5 gD ORF sequences (reported here) identified several amino acid mismatches. Since the MAbs 3402 and R54 neutralize the BHV-1 and not BHV-5, it was presumed that these were important amino acids in defining the epitope. To further localize the neutralizing epitopes, synthetic peptides corresponding to these regions in the BHV-1 gD ORF were tested for their capacity to block monoclonal antibody neutralization of BHV-1 infectivity. The peptides encompassing amino acids 92—106 (3402 epitope) and amino acids 202–213 (R54 epitope) of the BHV-1 gD competed with BHV-1 for the binding by MAbs 3402 and R54, respectively, in a dose-dependent manner. Antisera produced in rabbits to these peptides conjugated to a carrier reacted strongly with a 30-kDa protein by Western blotting and had neutralizing antibody titers against BHV-1.

We have previously prepared rabbit anti-idiotypic antibodies (anti-Ids) against the neutralizing monoclonal antibodies (MAbs) specific for the gp53 of bovine viral diarrhea virus (BVDV). The anti-Ids, purified by sequential immunoaffinity chromatography, inhibited the immunizing MAb from binding to the original antigens and blocked BVDV infection of cell cultures. This study evaluated immune responses in mice to the purified anti-Id reagents. BVDV-specific neutralizing antibodies were induced by the anti-Ids. The antisera (Ab3) induced by the anti-Ids immunoprecipitated gp53 from BVDV-infected Madin-Darby bovine kidney (MDBK) cell lysates. However, lymphocyte-proliferative responses were specific only for the respective immunizing antigens. These results suggest that the anti-Ids may bear an internal image of the gp53 to stimulate production of antibody but not to stimulate a virus-specific cellular immune response in mice.

Three experiments, using 85 crossbred beef calves, were conducted to evaluate the adjuvanticity of single, multiple, and combined doses of recombinant bovine IL-1 beta (rBoIL-1 beta) and recombinant bovine IL-2 (rBoIL-2), with a modified-live bovine herpesvirus-1/parainfluenza-3 (BHV-1/PI-3) virus vaccine and a killed bovine viral diarrhea (BVD) virus vaccine. Cytokines were administered intramuscularly at vaccination but at different injection sites. All cytokine treatments increased non-major histocompatibility complex (MHC)-restricted cytolytic capability of peripheral blood mononuclear cells (PBMC) against virus-infected target cells and serum neutralizing (SN) antibody titers to BHV-1 and BVD virus. Multiple, consecutive injections of rBoIL-2 generally showed the greatest adjuvant effect, and no additive effect was observed when rBoIL-1 beta and rBoIL-2 were administered together. In a challenge experiment, calves were vaccinated with a modified-live BHV-1/PI-3 vaccine and infected with BHV-1 on Day 21. Cytokine-treated calves had higher SN antibody titers to BHV-1 than did the control calves at the time of challenge. Calves that were administered rBoIL-2 on 5 consecutive days shed less BHV-1 and had the highest SN antibody titer to BHV-1 (Day 28). These data suggest that rBoIL-1 beta and rBoIL-2 may be useful immunoadjuvants for bovine respiratory disease virus vaccines.

We describe the preparation of monoclonal antibodies (Mabs) directed against recombinant bovine interleukin-1 beta (rBoIL-1 beta). These anti-IL-1 beta Mabs were designated SA10, SA12, SA13, SA15, and SA22, and were characterized on the basis of their epitope specificity and cross-reactivity with homologous and heterologous cytokines in enzyme-linked immunosorbent assays and immunoblot analyses. Additionally, the ability of these Mabs to neutralize IL-1 beta was tested in thymocyte costimulation assays. The ELISA titers of all Mabs ranged from 9.4 x 10(6) to 1 x 10(7). Data indicate that Mabs SA10, SA12, SA15, and SA22 neutralized both bovine macrophage-derived IL-1 (1:4) and rBoIL-1 beta (1 ng ml-1). All the Mabs against rBoIL-1 beta (SA10, SA12, SA13, SA15, SA22) were specific and did not cross-react with other cytokines tested, except recombinant human IL-1 beta (rHuIL-1 beta). This finding suggests that these Mabs recognize epitopes common to human and bovine IL-1 molecules. Competition experiments suggested that Mab SA22 recognized a different epitope and Mabs SA10, SA12, SA13, and SA15 recognized the same epitope on the rBoIL-1 beta molecule. These observations suggest that these Mabs could be useful reagents for developing immunoassays to measure bovine IL-1 beta from biological fluids and to study the immunoregulatory role of IL-1 in the bovine immune system.

A panel of murine monoclonal antibodies (MAbs) to bovine herpesvirus-1 (BHV-1) was prepared. Three of them were neutralizing MAbs and reacted against 130/75/50 kDa, 77 kDa, or 97 kDa glycoproteins (gp). A fourth non-neutralizing MAb recognized the 97 kDa gp. Competition radioimmunoassay demonstrated that each of the four MAbs reacted against a different virus epitope. Anti-idiotypic antibodies (anti-id) to the four MAbs were produced in rabbits and purified by sequential immunoaffinity chromatography. Each anti-id inhibited the binding of its respective MAb to BHV-1 in competitive ELISA and blocked BHV-1 neutralizing activity of the MAb. This inhibition suggested that the anti-ids were specific for the antigen binding site of the MAbs. Treatment of MDBK cells with anti-ids inhibited BHV-1 infection, which suggested that the anti-ids block a cellular component essential for virus infection. Absence of significant cross-reactivity among the anti-ids for heterologous MAbs indicated that they recognized unique determinants on the antigen binding site of the homologous MAb.

Four monoclonal antibodies (mAbs) (9.49, 24.27, 46.71 and 179.57) were produced against Fasciola hepatica excretory-secretory products. Isotype analysis revealed the antibodies to be IgM, IgG3, IgG1, and IgM. In immunoblot assays, the mAbs recognized different antigenic polypeptides migrating between 29 and 180 kDa. Specificity of the mAbs was evaluated by ELISA against antigens of Fascioloides magna, Anoplocephala magna, Stichorchis subtriquetrus, Haemonchus contortus, sheep liver extract (SLE), bovine liver extract (BLE), bovine serum albumin (BSA), bovine viral diarrhea virus (BVDV), and Madin-Darby bovine kidney (MDBK) cells. Monoclonals 9.49 and 24.27 were specific, and reacted only with Fasciola hepatica antigens. However, mAb 46.71 cross-reacted with antigens of Fascioloides magna, A. magna, Stichorchis subtriquetrus, and H. contortus but not with SLE, BLE, BSA, BVDV or MDBK cells. Monoclonal antibody 179.57 cross-reacted with Fascioloides magna, A. magna, S. subtriquetrus, H. contortus, SLE, and BLE, but not with BSA, BVDV, or MDBK cells.

Polyclonal rabbit anti-idiotypic antibodies (anti-ids) against two neutralizing murine monoclonal antibodies (mAbs) specific to a bovine viral diarrhea virus (BVDV) glycoprotein. 53 kDa, were produced, purified, and characterized. Each anti-id inhibited the binding of its respective mAb to BVDV antigen in a competitive ELISA and blocked the immunoprecipitation of the 53 kDa protein by the mAb. The anti-ids also inhibited the virus-neutralizing activity of their homologous mAbs. These results suggest that the anti-ids bear an internal image of a BVDV antigen and mimic neutralizing epitopes on the 53 kDa protein. Treatment of MDBK cells with the anti-ids inhibited BVDV infection, indicating that they block a cellular component, such as a virus receptor, required for virus adsorption or entry. Inhibition of the homologous mAb and lack of inhibition of the heterologous mAb indicate that the anti-ids are specific for the unique antigen-binding sites on the mAbs.

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are closely related bovine lentiviruses that are difficult to distinguish by presently available diagnostic methods. Recently, in our laboratory, a monoclonal antibody (MAb; MAb 10H1) against the BIV Gag protein identified a differential epitope, located at the 6.4-kDa N terminus of a 29-kDa Gag capsid protein, which was absent in JDV. To define the essential amino acids of the epitope, a series of primers within the 163 bp of DNA corresponding to the 6.4-kDa protein were designed. The full-length 163-bp DNA fragment and the smaller DNA fragments with deletions were amplified by PCR and then cloned into pQE32 vectors for protein expression studies. The expressed proteins were analyzed with MAb 10H1 by Western blotting. The differential epitope has been narrowed to a 26-amino-acid region (R121 to R146), which includes 6 residues of p16 MA (where MA represents the matrix protein) and 20 residues of p2L. A synthetic peptide corresponding to the putative 26-amino-acid epitope blocked MAb 10H1 binding to the expressed peptide. These experiments revealed that the epitope spans the cleavage site between p16 MA and p2L and presumably will be valuable in distinguishing the two viruses.

The monoclonal antibody (MAb) D89 against bovine viral diarrhea virus (BVDV) was used in conjunction with fluorescein-conjugated anti-mouse immunoglobulin in an indirect fluorescent antibody (IFA) procedure on frozen tissue sections and cell culture. During the 2-year study, BVDV was isolated from specimens submitted in 460 cases. The D89 Mab detected all but 2 BVDV isolates, both cytopathic. In 316 of the cases in which BVD virus was detected by IFA, specimens were inoculated on bovine turbinate cells and examined for BVDV antigens at 3–5, 10, and 20 days postinoculation. The BVDV was detected in 238/316 cases (75%) after 3–5 days incubation. The remainder were not detected until 10 or 20 days postinoculation. Virus isolation was enhanced in the early test if plates were centrifuged at the time of inoculation. Results suggest that D89 monoclonal antibody is a suitable diagnostic reagent for the detection of BVDV isolated from diagnostic specimens. The D89 MAb can be used for the detection of BVDV in both cell culture and tissues. Combination of D89 with another BVDV MAb (C 17) did not improve the ability to detect BVDV in tissues compared to using D89 only, and the combined Mab's resulted in an increase in nonspecific fluorescence when used on tissues. Although pooling of different BVDV monoclonal antibodies may be necessary to detect all strains of BVDV in cell culture, pooling should be used with caution on tissues. Early detection of BVDV in cell culture by this IFA procedure permits faster confirmation of BVDV diagnosis when compared to the usual routine testing for noncytopathic BVDV at termination of first passage in cell culture.

The National Animal Disease Laboratory (NADL) vaccine strain of bovine viral diarrhea virus (BVDV) genes for gp48 and p80 were expressed in Escherichia coli. The BVDV-NADL gene for gp62 was integrated into a baculovirus genome for expression in Spodoptera frugiperda (Sf-9) insect ovarian cells. The antigenicity of baculovirus expressed BVDV protein was detected by anti-BVDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent assay (IFA) and radio-immunoprecipitation (RIP). The recombinant proteins isolated from bacteria showed antigenic properties when analyzed by ELISA and immunoblotting using BVDV antibodies. The recombinant proteins were then used in ELISA or IFA to detect BVDV infection by testing 54 independent bovine serum samples. The baculovirus-expressed BVDV protein was used as an ELISA and IFA antigen, and the bacteria-expressed proteins were used as ELISA antigens. BVDV-NADL-infected Madin-Darby bovine kidney (MDBK) cell monolayers served as a control antigen. Statistical analysis showed a high degree of correlation between the reactivity of recombinants and natural antigens in ELISA using bovine sera. The results of ELISA or IFA proved there is a high degree of correlation with the virus neutralization. In the comparative ELISA assays, the insect-cell-mediated expression revealed greater specificity and sensitivity than the bacterial expression or the natural BVDV antigens produced by cell cultures.

The present study was designed to evaluate the effect of bovine immunodeficiency virus (BIV) infection on immune functions and possible interactions between BIV and other bovine viruses in calves. Ten calves were inoculated intravenously with BIV, and five served as controls. An increased lymphocyte proliferation to BIV gag protein was demonstrated 2 to 6 weeks after BIV inoculation (P < 0.05). Lymphocyte subset differentiation revealed a decreased CD4/CD8 ratio (P < 0.05) during weeks 2 to 7, suggesting a possible immune dysfunction in BIV-infected calves. When the calves were inoculated with bovine herpesvirus type 1 (BHV-1), the antibody response to BHV-1 in BIV-infected calves was delayed and the antibody titers were significantly lower (P < 0.05). Injection of bovine viral diarrhea virus vaccine also elicited a lower neutralizing antibody response in BIV-infected calves. The results indicated that immune suppression occurred in BIV-infected calves.

Bovine immune responses to rabbit antiidiotypic antibodies (anti-Id) against neutralizing monoclonal antibodies to bovine herpesvirus-1 (BHV-1) envelope glycoproteins and to BHV-1 glycoproteins were compared. Glycoprotein-immunized animals produced high titers of anti-BHV-1 antibodies and were protected against BHV-1 challenge. Recombinant bovine interleukin-2 (rIL-2)-treated, anti-Id-immunized animals showed a slight reduction in clinical disease, and one calf produced BHV-1-neutralizing antibodies. Treatment with rIL-2 augmented non-BHV-1-specific immune responses. However, even with rIL-2 as an adjuvant, the mixture of polyclonal anti-Id did not elicit a consistent, protective BHV-1-specific immune response in calves.

Bovine viral diarrhea (BVD) virus inhibited phytohemagglutinin (PHA)-, PHA plus phorbol-12-myristate-13 acetate (PMA)- or PHA plus calcium ionophore (A23187)-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation. Further, BVD-virus inhibited A23187-stimulated leukotriene B4 (LTB4) synthesis into the culture supernatants. Presence of exogenous LTB4 failed to reverse the BVD virus-induced immunosuppression. Our results suggest that BVD virus-induced immunosuppression is due to a factor that may be necessary to induce LTB4 synthesis for normal mononuclear cell proliferation.

Previously, we prepared rabbit anti-idiotypic (anti-Id) antibodies against murine monoclonal antibodies (MAbs) specific for the major bovine herpesvirus-1 (BHV-1) envelope glycoproteins. Glycoprotein III (gIII) contains neutralization epitopes and may be the virus attachment protein. Anti-Id antibodies to a neutralizing MAb that reacts with gIII were purified by sequential immunoaffinity chromatography. Immune responses to the purified anti-Id reagent and BHV-1 were compared in mice. Both groups of mice produced BHV-1-specific neutralizing antibodies. However, lymphocyte proliferative responses and interferon and interleukin-2 production were specific for the respective immunizing antigens. These results suggest that the anti-Id reagent may bear an internal image of a B-cell-stimulating epitope of glycoprotein gIII; however, this epitope does not stimulate a virus-specific cellular immune response in mice.

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a potent and selective inhibitor of protein kinase C (PKC), inhibited PHA-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation, interleukin-2 (IL-2) production, and cytosolic PKC activity without affecting the cell viabilities. Presence of exogenous cytokines, such as purified human IL-2 or recombinant bovine IL-2 (rbovIL-2), reversed the H-7 inhibitory effects on PHA-stimulated PBMC proliferation. We conclude that the PKC enzyme plays an important role as a second messenger in bovine PBMC proliferation in the early stages of cell activation.

Bovine viral diarrhea (BVD) virus inhibited phytohemagglutinin (PHA)-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation and bovine interleukin-2 (IL-2) production. In the controls, the heat-inactivated BVD virus was not capable of suppressing the PHA-stimulated PBMC proliferation. Presence of exogenous cytokines, such as purified human IL-2, recombinant bovine interleukin-1 (rbovIL-1), recombinant bovine IL-2, and recombinant human IL-6 failed to reverse the BVD virus-induced immunosuppression. Also, we found that the BVD virus inhibited PHA and IL-2 induced proliferation of bovine PBMC in the early and late stages of activation. In summary, our data suggest that BVD virus induced immunosuppression was not due to destruction of the PBMC but may be inhibiting one or more of the important intracellular enzymes that may regulate PBMC proliferation.

Infectious bovine rhinotrachetis virus (IBRV) progeny was increased ten to 12 fold in bovine turbinate (BT) cells treated with 10 −3 M corticosterone acetate (CCA) as demonstrated by plaque assay. Autoradiographic studies demonstrated an increased binding of 3 H-corticosterone ( 3 H-CS) in IBRV infected cells and the fractionation of labelled cells revealed 78–80% of the total hormone associated with the cytoplasmic components. Incorporation of 3 H-uridine and 3 H-valine precursors into cells treated with the hormone demonstrated up to 16-fold increase in RNA and protein synthesis which was inhibited by the addition of actinomycin D. The data suggest that increased rate of macromolecular synthesis in IBRV infected cells treated with the corticosteroid may result in the enhancement of virus production.

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.

Hemagglutination activity, structural protein profiles and neutralization assays were used in a comparative study of bovine herpesvirus 1 strains from the U.S.A., Canada, Great Britain, Denmark and Malaysia with equine, feline and human herpesviruses in order to further characterize the bovine herpesvirus 1 hemagglutinin. Bovine herpesvirus 1 strains of different geographical origins all showed hemagglutinating activity for mouse erythrocytes; furthermore, feline herpesvirus 1 was also shown to hemagglutinate mouse erythrocytes. Analyses of partly purified viruses showed that a distinctive and specific polypeptides profile is associated with each species of herpesviruses used in our study; strains of bovine herpesvirus 1 from North America, Europe and Southeast Asia however, presented a remarkable similarity as to their electrophoretic protein patterns. A protein similar to the 97-kDa bovine viral hemagglutinin was not identified with the hemagglutinating feline herpesvirus. An important neutralization epitope on the bovine viral hemagglutinin was also not found on feline, equine and human herpesviruses but was identified on all bovine strains tested from North America, Europe and Southeast Asia stressing the importance of the bovine hemagglutinin for eventual prophylactic purposes.

Twenty-eight Holstein heifer calves were allotted at birth to one of four treatments: 1) 0 mg, 2) 1,400 mg, or 3) 2,800 mg of dl-alpha-tocopherol acetate given orally at weekly intervals, or 4) 1,400 IU of dl-alpha-tocopherol weekly by intramuscular injection in order for us to study their performance and metabolic profile. Calves were fed milk at 8% of birth weight until they were weaned at 6 wk of age and fed a complete calf starter ad libitum from birth. Calves were on experiment for 12 wk. There were no significant differences in weekly weight gains, starter consumption, and fecal scores among treatments. However, there was a trend toward greater starter consumption and weight gains in supplemental calves. Serum alpha-tocopherol concentration measured after 7 d of each administration was significantly higher at wk 4 in calves given the high oral supplementation and at wk 2, 4, 6, and 8 higher in injected calves than in unsupplemented calves. Creatine kinase activity was higher in unsupplemented calves and negatively correlated with serum alpha-tocopherol until wk 8, suggesting preclinical muscular dystrophy. Alkaline phosphatase activity was higher with the high oral supplementation. Serum carbon dioxide values showed a trend toward positive correlation with those for serum tocopherol; however, the values were within normal range. There were no significant differences in creatinine, glucose, phosphorus, calcium, urea nitrogen, chloride, sodium, potassium, albumin, and total protein among treatments. Serum glucose was higher in all calves at wk 10 and 12 than at wk 4, 6, and 8. Calves may not get enough vitamin E with conventional calf starters, and supplementation may be essential to obtain maximum performance.

Thirty-two Holstein heifer calves, eight per group, were fed 0, 125, 250 or 500 IU/d of supplemental vitamin E/calf, from birth to 24 wk of age, in order to determine the effect on their immune responses. Overall mean lymphocyte blastogenic responses to various T-cell and B-cell mitogens were higher in supplemented calves than in control calves. Mean concentrations of cortisol in serum were lower in all supplemented calves than in control calves. Antibovine herpes-virus type 1 antibody titer (IgG) at 8 and 9 wk, in response to a commercial modified-live intranasal vaccine at 7 wk, was similar in all treatment groups. At 24 wk, in response to a booster at 21 wk, titer was higher in calves given 125 IU of vitamin E/d than in control calves. Based on the concentrations used, it is concluded that supplementation of conventional rations with 125 IU of vitamin E/d may maximize immune responses in calves and may be cost effective.

Bovine viral diarrhea virus (BVDV) antigen was detected in cell culture with an indirect immunoperoxidase (IP) procedure using a specific monoclonal antibody, and an avidin-biotin-peroxidase complex. Cytopathic and noncytopathic strains of the virus showed similar patterns of IP staining until 3 days post-infection. At six days post-infection, intensity of staining decreased in cell cultures infected with noncytopathic virus, but not with cytopathic virus. The IP procedure detected BVDV antigen in cells used to isolate virus from tissues of aborted bovine fetuses and peripheral blood lymphocytes of adult cattle. Immunoperoxidase detected BVDV isolates from 10 of 44 cases of abortion of which seven of these were noncytopathic. Noncytopathic BVDV isolates from the peripheral blood lymphocytes of 7 of 65 animals were identified.

The effect of vitamin E on immune responses of Holstein calves was investigated. Treatments were: 0, 1400, and 2800mg of dl- α -tocopheryl acetate given orally at weekly intervals or 1400mg of dl-α-tocopherol weekly by injection. Calves were fed milk for 6 wk and then fed a complete calf starter ad libitum. Calves were on experiment until they were 12 wk of age. Lymphocyte stimulation indices were significantly higher for calves given the high amount of oral supplementation and for injected calves than for unsupplemented calves. There were no significant differences at any of the individual weeks between unsupplemented and orally supplemented calves. Injected calves showed significantly higher values than unsupplemented calves at wk 4 and than all other calves at wk 8. There were no significant differences in the concentrations of immunoglobulins G 1 and G 2 among treatments. Immunoglobulin M was significantly higher at wk 6 in calves given the high amount of oral supplementation than in all other calves. At wk 12, serum from calves given the high amount of oral supplementation and calves given injections inhibited infectious bovine rhinotracheitis viral replication in tissue cultures as compared with those of unsupplemented calves. In supplemental experiments serum α-tocopherol and lymphocyte stimulation indices of yearling heifers determined 7 d after a single injection of 2000 IU of dl-α-tocopherol were significantly higher than preinjection values. In vitro addition of vitamin E to lymphocyte cultures did not increase phytohemagglutinin-induced blastogenesis.

Intracellular virus-induced polypeptides from 3 cytopathogenic and 2 non-cytopathogenic bovine viral diarrhea (BVD) virus reference strains were analyzed by radioimmunoprecipitation and polyacrylamide gel electrophoresis, using a specific bovine multivalent antiserum and a neutralizing BVD-virus monoclonal antibody. Electrophoretic patterns of major proteins demonstrate extensive variation between strains. Most notably, a major 80,000 (80K) polypeptide was present in all cytopathogenic strains but absent in both non-cytopathogenic strains. Furthermore, a neutralizing monoclonal antibody produced against the NADL strain immunoprecipitated a 53K glycoprotein indicating that this protein carries an important neutralization epitope that is not present in all strains tested.

Peripheral blood mononuclear cells obtained from 4- to 6-month-old-calves were inoculated in vitro with bovine herpesvirus-1, parainfluenza-3, or bovine virus diarrhea viruses. No increase in infectious virus progeny was observed; however, the viruses were detected in the cells for at least 96 h post-infection without any significant reduction in cell viability. The three viruses, either alone or in combination, suppressed phytohemagglutinin-induced proliferation of the mononuclear cells. The greatest suppression was observed in cultures inoculated with bovine virus diarrhea virus. Addition of isoprinosine partially restored this viral-induced suppression of proliferative response, and the efficiency of reversal was greater in bovine virus diarrhea virus-infected cells. Interleukin-2 activity was higher in cultures of virus-infected mononuclear cells than in cultures of non-infected cells.

Twenty-five calves were allotted to five groups: controls that did not receive bovine recombinant interleukin-2 (rIL-2) and four groups that received 5 daily injections of rIL-2 at 11.4, 1.1,0.11, or 0.0 II ~g/Ib/day. On day 0 of the experiment, all calves received bovine herpesvirus-I (BHV-1) vaccine and the first of the 5 daily injections of bovine rIL-2. All calves were infected with BHV-Ion day 21 of the experiment. Calves treated with 11.4 ~g/Ib/day had elevated rectal temperatures and mild diarrhea during administration of rIL2. All other calves were normal. Compared to control calves, those treated with 11.4, 1.1, and 0.11 ~g/Ib/day had higher (P