Patterns of bovine T cell-mediated immune responses to bovine herpesvirus 1

Lymphocyte proliferation was used to evaluate T cell-mediated immune responses to different isolates of Bovine Herpesvirus 1 (BHV-1). Groups of high, moderate, and low responses were observed when lymphocytes from three breeds of dairy cattle were stimulated with each of the BHV-1 isolates. Proliferation of cells in the low responding group could be augmented by exogenous IL-2. The mechanism of unresponsiveness by cells from one individual whose response was not altered by IL-2 supplementation was further investigated. The patterns of response by limiting dilution frequency analysis eliminated the possibility that this individual lacked responsive cells but suggested the presence of regulatory cell interactions which resulted in the observed low proliferative response. These results show that animals exposed to the same environment can vary greatly in their ability to respond to BHV-1. At least two mechanisms may be responsible for low proliferative responses in vitro: inadequate levels of IL-2 and the presence of suppressor cells.     

Prevalence of bovine herpesvirus 1 (BHV-1) infection in Hungarian cattle herds.

Hungarian cattle herds were surveyed for bovine herpesvirus 1 (BHV-1) infection by ELISA of milk and serum samples. In 1993, 75% of the large cattle herds (consisting of more than 50 cattle) and all small herds (small-scale producers' stocks), while in 1997 90% of the small herds were included in the survey. In the case of large herds, 79.3% of the herds and 64.1% of the samples tested were found to be positive. Of the small herds, 13.5% and 15.7% tested positive in 1993 and 1997, respectively. The majority of large herds were Holstein-Friesian dairy stocks. Small herds with an infection rate markedly exceeding the average were found in those counties where the small herds had been in close contact with the large-scale farms, or where new herds were established by using animals of uncontrolled infectious bovine rhinotracheitis (IBR) status originating from large farms. Attention is called to the importance of maintaining the IBR-free status of small herds that constitute one-third of the Hungarian cattle population.     

Humoral and T cell-mediated immune responses to bivalent killed bovine viral diarrhea virus vaccine in beef cattle

The objective of this research project was to evaluate the antibody and cell-mediated immune responses to a multivalent vaccine containing killed bovine viral diarrhea virus (BVDV) types 1 and 2. Twenty castrated male crossbred beef cattle (350-420kg body weight) seronegative to BVDV were randomly divided into two groups of 10 each. Group 1 served as negative mock-vaccinated control. Group 2 was vaccinated subcutaneously twice, 3 weeks apart, with modified live bovine herpesvirus 1, parainfluenza 3 virus and bovine respiratory syncytial virus diluted in diluent containing killed BVDV type 1 (strain 5960) and type 2 (strain 53637) in an adjuvant containing Quil A, Amphigen, and cholesterol. Serum samples were collected from all cattle at days -21, 0, and days 21, 28, 35, 56 and 70 post-vaccination. Standard serum virus neutralization tests were performed with BVDV type 1 (strain 5960) and type 2 (strain 125C). Anticoagulated blood samples were collected at day 0, and days 28, 35, 56 and 70 post-vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated, stimulated with live BVDV type 1 (strain TGAN) and type 2 (strain 890) and cultured in vitro for 4 days. Supernatants of cultured cells were collected and saved for interferon gamma (IFNgamma) indirect enzyme-linked immunosorbent assay (ELISA). Four-color flow cytometry was performed to stain and identify cultured PBMC for three T cell surface markers (CD4, CD8, and gammadelta TCR) and to detect the activation marker CD25 (alpha chain of IL-2 receptor) expression. The net increase in %CD25+ cells (Delta%CD25+) of each T cell subset of individual cattle was calculated. The results of all post-vaccination weeks of each animal were plotted and the areas under the curve of each T cell subset were statistically analyzed and compared between groups. The mean area under the curve of the Delta%CD25+ data for days 0-70 of all subsets, except CD4-CD8+gammadelta TCR- (cytotoxic) T cell subset of both BVDV types 1 and 2 stimulated cells, of the vaccinated group were significantly higher than the control group (P<0.05). IFNgamma production by PBMC from the vaccinated group showed significantly higher results (P<0.05) than the control group in the BVDV types 1 and 2 stimulated cells for at least some time points after vaccination. The vaccinated group also had significantly (P<0.0001) higher neutralizing antibody titers than the control group from day 28 onward.     

A bovine herpesvirus (BHV-4) as passenger virus in ethmoidal tumours in Indian cattle

A herpesvirus was isolated from tumours of the ethmoidal mucosa in two of three head of cattle in the State of Kerala, India. The virus designated M40 was cytopathic for a variety of cultured bovine and porcine cells and it did not kill suckling mice or chicken embryos. Sera from tumour-bearing cattle and goats reacted with the M40 virus. Immunofluorescence tests with FITC-conjugated IgG from a bovine monospecific antiserum to bovine herpesvirus 4 (BHV-4) stained the M40 virus specific antigen in infected cells. Experimental infection of goats with the M40 virus did not result in development of tumours. This virus is therefore considered to represent a "passenger" virus. A great similarity was found between restriction patterns of DNAs extracted from M40 virus and the strain 66-P-347, a reference strain of the BHV-4 group.     

Bovine herpesvirus type 1 (BHV-1) up-regulates telomerase activity in MDBK cells

The proliferative capacity of mammalian cells is regulated by telomerase, an enzyme uniquely specialised for telomeric DNA synthesis. The critical role of telomerase activation in tumor progression and maintenance has been well established in studies of cancer and of oncogenic transformation in cell culture. Experimental data suggest that telomerase activation has an important role in normal somatic cells, and that failure to activate sufficient telomerase also promotes disease. Evidence regarding the role of telomerase in the pathogenesis of several viruses including human immunodeficiency virus has led to an increased interest in the role of telomerase activity in other virus infections. In this research we evaluated the telomerase modulating activity of Bovine herpesvirus 1 (BHV-1) in MDBK cells. MDBK cells were infected at different multiplicity of infection with BHV-1 Cooper strain and telomerase activity at different times post-infection was measured by the TRAP assay. Our data indicate that BHV-1 significantly up-regulates telomerase activity at 3 and 6h post-infection decreasing after the 24h post-infection. Our data, showed that the effect was mediated by an immediate-early or early viral gene, and use of the protein translation inhibitor cycloheximide confirmed that an immediate early gene is primarily responsible.     

Antiapoptotic activity of bovine herpesvirus type-1 (BHV-1) UL14 protein

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. One previous study showed that transient transfection of bovine herpesvirus type-1 (BHV-1) UL14 protein is efficient in protecting Madin Darby kidney (MDBK) and human chronic myelogenous leukemia (K562) cells from sorbitol-induced apoptosis. This protein corresponds to a putative protein of BHV-1, which shares aminoacid sequence with a part of the peptide-binding domain conserved in human heat shock protein (HSP70) family. The pBK-CMV-UL14 plasmid transfected MDBK cells treated with sorbitol did not show caspase-3 and caspase-9 activation with respect to non-transfected MDBK cells (UL14 negative). Furthermore, we report that the expression of the full length sequence of BHV-1 UL14 is evident after 7 h of infection of BHV-1 on MDBK cells which were then treated with sorbitol. These results indicate that UL14 gene product has important implications to enhance cell survival in response to apoptotic stimuli.     

Effect of age and pregnancy on the antibody and cell-mediated immune responses of horses to equine herpesvirus 1.

The cell-mediated immune response and antibody response of horses of varying ages and of pregnant horses to equine herpesvirus 1 antigen were examined. Six to eight month old horses showed either no increase or slight increases in anti-equine herpesvirus 1 serum neutralizing antibody following vaccination and revaccination with a modified live equine herpesvirus 1 vaccine. However, these same horses showed a marked increase in the cell-mediated immune response to equine herpesvirus 1 as measured by the lymphocyte transformation test. Eighteen to 21 month old horses showed four to 64-fold increases in anti-equine herpesvirus 1 serum neutralizing antibody titer following vaccination, but the cell-mediated immune response to equine herpesvirus 1 was low or absent. Only after revaccination did they show an increased cell-mediated immune response to equine herpesvirus 1. The cell-mediated immune response of mares in the latter stages of pregnancy to equine herpesivurs 1 was suppressed although antibody titers increased as much as 16-fold following exposure to virulent equine herpesvirus 1.     

BHV-1 vaccine induces cross-protection against BHV-5 disease in cattle

Protection against BHV-5 disease induced by inactivated BHV-1 or BHV-5 based vaccines was analysed. Two groups of calves were subcutaneously immunized with an inactivated BHV-1 or BHV-5 based vaccine. A third group was not vaccinated and used as control. In the post-vaccination period, we studied the humoral and cellular immune response resulting similar to both groups. The efficacy of the vaccines was tested after intranasal challenge of the calves with a virulent Argentinean BHV-5 isolate (A-663). All control animals developed neurological signs associated with BHV-5 infection and high levels of virus shedding. Calves immunized with the BHV-1 and BHV-5 inactivated vaccines were protected against BHV-5 disease. Our study provides evidence that strongly support the existence of cross-protection between BHV-1 and BHV-5 in calves. Even though this has already been suggested by previous works, this is the first time an exhaustive study of the immune response is performed and typical clinical BHV-5 meningoencephalitis signs are reproduced in an experimental BHV-5 challenge trial.     

Temporal cell-mediated immune responses of cattle following experimental and natural exposure to living Brucella abortus.

A study on cell-mediated immune responses in cattle with different exposure experiences to Brucella abortus was conducted by an in vitro lymphocyte stimulation assay. The purpose of this study was to determine how soon the cell-mediated immune responses would be detected following experimental exposure to B. abortus and to study the cell-mediated immune trend following experimental and natural exposure of cattle to B. abortus. The first positive cell-mediated immune responses occurred one to two weeks after experimental inoculation with living B. abortus strain 2308. The cell-mediated immune responses in these animals appeared at least one week before the appearance of of B. abortus serum agglutinating antibodies. Animals which were naturally infected with B. abortus biotypes 1 and 2 demonstrated positive cell-mediated immune responses throughout the study.     

Restriction of bovine herpesvirus 1 (BHV-1) growth in non-permissive cells beyond the expression of immediate early genes

Mouse BALB/3T3-A31-1-1 (A31) cells are non-permissive to bovine herpes virus-1 (BHV-1) but permissive to pseudorabies virus (PrV). The promoter activity of the immediate early gene of BHV-1 (BICP4) was very weak when compared with that of PrV in A31 cells. Infectious BHV-1 genomic DNA co-transfected into A31 cells with plasmids expressing BICP4 and BICP0 by a strong promoter failed to yield any progeny virus. Growth of BHV-1 in non-permissible A31 cells is restricted in many phases of the growth. The fact that expression of BICP4 and/or BICP0 in A31 cells does not improve the yield of progeny virus from infectious BHV-1 genomic DNA suggests that some more growth restrictions exist beyond the expression of BHV-1 immediate early proteins.     

The epizootology of caprine herpesvirus (BHV-6) infections in goat populations in Greece

Previous preliminary studies had shown that caprine herpesvirus (BHV-6) infections exist in many countries where goats play an economical role. The extensive serum survey made in Greece reveals that the virus must be widespread because more than 50% of the goats have antibodies. The bucks were found to have a higher evidence of infections than the female goats. The kids possessing maternal antibodies became seronegative at the age of 4 months and new antibodies appeared at the age of 7–8 months. Observations in 2 closed goat herds showed that the virus does not spread during the mating and lactating period. Neutralization titres increased or new infections in seronegative animals occurred after the summer when breeding took place. The virus is present in a latent state and recurrent infections are assumed to occur. In spite of that all our efforts to isolate the virus were unsuccessful, even after experimental immunosuppression.     

Molecular Typing of a BHV-4 (Bovine herpesvirus 4) Field Isolate

A new BHV-4 (bovine herpesvirus 4) isolated from a case of bovine interdigital dermatitis was characterized by PCR and restriction enzyme analysis. To determine whether the new isolate (PR/1) belonged to BHV-4, DNA from infected cells was specifically amplified by PCR. We used a set of primers spanning a large 2.571 kb conserved region of the BHV-4 genome, including the 3 end of ORF1 (homologous to the EBV BVRF1 gene), ORF2 (homologous to the EBV BXRF1 gene), ORF3 (TK gene) and ORF4 (gH gene) 5 end, respectively. The identity of the amplified product was confirmed by HindIII restriction enzyme digestion and Southern hybridization. No product was observed from the DNA of other bovine herpesviruses tested. The restriction patterns of the PR/1 genome compared to DN 599, MOVAR 33/63 and LVR BHV-4 reference strains showed two kinds of differences, either related or not related to the prDNA (polyrepetitive DNA). Taken together, these data show that PR/1 is a new BHV-4. We would consider that the present report provides a scheme of work for diagnosis and typing of BHV-4 isolates.     

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.     

Equine cell-mediated immune response to Rhodococcus (Corynebacterium) equi

A lymphocyte blastogenic assay was developed to serve as an in vitro correlate of cell-mediated immunity to Rhodococcus (Corynebacterium) equi (R equi) in the equine species. Lymphocytes obtained from a group of experimental ponies showed no response in cell culture to R equi heat extract or lysozyme extract antigens. Ponies were assigned to groups for experimental inoculation. Three ponies were inoculated subcutaneously with live R equi, 3 were given live R equi by intranasal and intratracheal routes, and 4 ponies were left untreated. Lymphocytes from all inoculated ponies had a mitogenic response to R equi antigens in lymphocyte blastogenic assays performed between the 7th and 40th days after inoculation. Lymphocytes from noninoculated control ponies remained unresponsive to R equi antigens. Delayed-type hypersensitivity reactions developed in all experimentally exposed ponies after intradermal administration of the R equi antigen preparations. In a 2nd phase of experimentation, blastogenesis assays were performed on lymphocytes from horses in herds with endemic R equi infections. Results indicated that many of the animals had significant (stimulation index greater than 2) cell-mediated responses to the bacterium, but there was no distinct correlation between the immune response and clinical history. These data indicated that cell-mediated immunity is involved in the interaction of the equine immune system with R equi.     

Detection of antibodies to bovid herpesvirus 2 infection of cattle in Syria

Neutralising antibody to bovid herpesvirus 2 was demonstrated in the serum of 31 (10.8%) of 286 heads of cattle in north, south, and west Syria. 38% of titres were 1:2 to 1:8. There is no published report on isolation of this virus in Syria.