Isolation of a glycoprotein E-deleted bovine herpesvirus type 1 strain in the field

During a field trial to evaluate the efficacy of repeated vaccinations with bovine herpesvirus type 1 (BHV-1) marker vaccines, a glycoprotein E (gE)-negative BHV-1 strain was isolated from the nasal secretions of two cows, eight months after vaccination with a gE-negative live-attenuated vaccine, initially given intranasally, then intramuscularly. The strain isolated was characterised using immunofluorescence, restriction analysis and PCR. All the techniques used identified the isolated virus as a gE-negative BHV-1 phenotypically and genotypically identical to the Za strain used as a control.     

Evaluation of an immunofluorescent antibody test to detect bovine herpesvirus 1-specific IgM.

An indirect immunofluorescent antibody test (IIFAT) was developed to detect bovine herpesvirus 1 (BHV-1)-specific IgM. All sera were treated with protein-G agarose prior to testing to eliminate the possibility of false-positive results due to IgM-isotype rheumatoid factor (IgM-RF). Specific IgM was first detected 8 days after experimental infection of 3 calves free of maternally derived antibody, with peak responses occurring 2-7 days later. Seroconversion was detected in all 3 calves using a single-dilution enzyme-linked immunosorbent assay. Following reinfection at 30 days postinfection, a low-level IgM response was detected in only 1 calf. Seroconversion was detected in 2 calves. There was no evidence of activation of IgM-RF by infection or reinfection with BHV-1. When 87 acute and convalescent serum pairs collected from 21 outbreaks of respiratory disease were tested, specific IgM was detected in 58 animals (66.6%) from 19 (90.5%) outbreaks. Seroconversion was detected in 44 of these animals (50.6%) from 17 outbreaks (81.0%). The correlations between these 2 assays on a calf and outbreak basis were 79.3% and 90.5%, respectively. Specific IgM was detected in 17/20 sera (85.0%) collected from an additional outbreak. No virus was detected by virus isolation or immunofluorescent staining in nasal mucus samples collected at the same time. Detection of specific IgM by IIFAT is a useful technique for the serodiagnosis of BHV-1 infection.     

Analysis of bovine trigeminal ganglia following infection with bovine herpesvirus 1

Following primary infection of the eye, oral cavity, and/or nasal cavity, bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic (TG) neurons. Virus reactivation and spread to other susceptible animals occur after natural or corticosteroid-induced stress. Infection of calves with BHV-1 leads to infiltration of lymphocytes in TG and expression of IFN-gamma (interferon-gamma), even in latently infected calves. During latency, virus antigen and nucleic acid positive non-neural cells were occasionally detected in TG suggesting there is a low level of spontaneous reactivation. Since we could not detect virus in ocular or nasal swabs, these rare cells do not support high levels of productive infection and virus release or they do not support virus production at all. Dexamethasone (DEX) was used to initiate reactivation in latently infected calves. Foci of mononuclear or satellite cells undergoing apoptosis were detected 6h after DEX treatment, as judged by the appearance of TUNEL+ cells (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling). BHV-1 antigen expression was initially detected in lymphocytes and other non-neural cells in latently infected calves following DEX treatment. At 24h after DEX treatment, viral antigen expression and nucleic acid were readily detected in neurons. Our data suggest that persistent lymphocyte infiltration and cytokine expression occur during latency because a low number of cells in TG express BHV-1 proteins. Induction of apoptosis and changes in cytokine expression following DEX treatment correlates with reactivation from latency. We hypothesize that inflammatory infiltration of lymphoid cells in TG plays a role in regulating latency.     

Molecular cloning of DNA from a bovine herpesvirus 1 strain isolated in Hungary

Molecular cloning of the HindIII fragments of bovine herpesvirus 1 (BHV-1) strain HB144, isolated from infectious bovine rhinotracheitis (IBR) in Hungary, and of an infectious pustular vulvovaginitis (IPV) reference strain (K22) is reported. So far 52% of the IBR viral genome and 28% of the IPV viral genome have been cloned. The analysis of differences between the strains is currently in progress.     

Serological survey of bovine herpesvirus type 1 infection in China

To understand the nationwide seroprevalence of bovine herpesvirus type 1 (BoHV-1) infection of cows in China, 1344 sera of dairy cows from 29 provinces and 765 sera from 6 herds in Hubei province were collected with stratified random sampling. Another 483 sera from imported cows were included. The serum antibody was tested by BoHV-1 gG ELISA. The results demonstrated that the overall nationwide seroprevalence was 35.8% (481/1344), while the prevalence for individual province ranged from 12.1% to 77.8%. Although each province had positive samples, the prevalence was clustered in areas based on the cow population size. In Hubei Province, the overall seroprevalence was 22.2% (170/765) while the prevalence for individual farms varied greatly from 0.0% to 41.5%. The sera from imported cows had a moderate prevalence of 21.7% (105/483).     

A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions

We describe a rapid, sensitive and specific polymerase chain reaction (PCR) assay for the detection of BHV1 DNA in a range of routine diagnostic submissions without the need for prior virus isolation. The assay, which is based on the selected amplification of a portion of the viral tk gene, detected both BHV1.1 and BHV1.2 subtypes in a panel of 15 characterised field isolates, and its sensitivity was estimated to be <0.125 TCID(50). BHV2, alcephaline herpesvirus, BHV4, equine herpesvirus 1 (EHV1), EHV4 and pseudorabies virus were not detected confirming the specificity of the assay. One hundred and five diagnostic submissions, including tissues, nasal secretions and nasal swabs were taken from cattle with respiratory disease and tested using the routine methods of virus isolation (VI) and the fluorescent antibody test (FAT), and the results were compared with those obtained by PCR. The PCR assay detected BHV1 DNA in all samples that were positive by VI. BHV1 DNA was also detectable by PCR in raw and extended semen samples at a sensitivity of 1 TCID(50) per 50microl. The assay also detected BHV5, permitting differentiation between it and BHV1 by virtue of the size of the amplified PCR product. The PCR assay is more sensitive and independent of sample quality than either virus isolation or FAT, and it is faster than virus isolation. The sample preparation method is simple with few steps involved. There are no extra post-amplification blotting/hybridisation steps and the assay is not based on a nested PCR strategy that might otherwise exacerbate the problem of oversensitivity/contamination in the routine use of such a test in a diagnostic laboratory. This assay would permit discrimination between those animals naturally infected with wild type BHV1 and those vaccinated with tk-BHV1 strains.     

Diagnosis and control of Vibrio foetus infection in the bovine

Extract

During the past thirty years Vibrio foetus infection in cattle has been reported from most countries of the world. Sporadic abortions, usually in mid-gestation, have been accepted until recently as the most important aspect of the infection. Of late years, however, it has become apparent that V. foetus can be responsible also for poor conception rates-associated probably with early embryonic mortality. It has been shown further that V. foetus can be transmitted not only by the bull at the time of service but also in semen used for artificial in semi nation.     

Application of immunoperoxidase-based techniques to detect herpesvirus infection in tortoises.

Indirect (IIP) and direct (DIP) immunoperoxidase assays were developed for the serological and histological diagnoses of herpesvirus infection in tortoises, respectively. A mouse monoclonal antibody (MAb HL1546), specific for the heavy chain of tortoise IgY, was used as the secondary antibody in the IIP assay. Rabbit polyclonal antisera raised against 2 sucrose gradient-purified tortoise herpesvirus isolates (HV4295/7R/95 and HV1976) were used as primary antibodies for the detection of herpesvirus antigen either in infected cell cultures or in formalin-fixed, paraffin-embedded tissues. The IIP and DIP assays could detect either the presence of anti-herpesvirus antibody in the plasma of exposed tortoises or the presence of herpesvirus antigen in infected tissues, respectively. Although the IIP test complements the enzyme-linked immunosorbent assay and the serum neutralization test already available for measuring herpesvirus-specific antibody in tortoises, the DIP test is useful for the histological diagnosis of herpesvirus infection in tortoises.     

Encephalitis induced by bovine herpesvirus 5 and protection by prior vaccination or infection with bovine herpesvirus 1.

Calves were intranasally challenged with bovine herpesvirus 5 (BHV5) and followed for the development of viral infection, clinical encephalitis, histologic lesions in the brain, and viral sequences in the trigeminal ganglia. Calves that were previously vaccinated with bovine herepesvirus 1 (BHV1, n = 4) or previously infected with BHV1 (n = 5) or that had not been exposed to either virus (n = 4) were compared. No calf developed signs of encephalitis, although all calves developed an infection as indicated by nasal secretion of BHV5 and seroconversion to the virus. Histologic lesions of encephalitis consisting of multifocal gliosis and perivascular cuffs of lymphocytes were observed in calves not previously exposed to BHV1. BHV5 sequences were amplified from the trigeminal ganglia of calves previously vaccinated and from calves not previously exposed to BHV1; calves sequentially challenged with BHV1 and later BHV5 had exclusively BHV1 sequences in their trigeminal ganglia. Administration of dexamethasone 28 days after BHV5 challenge did not influence clinical disease or histologic lesions in either previously unexposed calves (n = 2) or previously immunized calves (n = 2), although it did cause recrudescence of BHV5, as detected by nasal virus secretion.     

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

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

Ruminant alphaherpesviruses related to bovine herpesvirus 1

Herpesviruses have mainly co-evolved with their hosts for millions of years. Consequently, different related host species may have been infected by various genetically related herpesviruses. Illustrating this concept, several ruminant alphaherpesviruses have been shown to form a cluster of viruses closely related to bovine herpesvirus 1 (BoHV-1): namely bovine herpesvirus 5, bubaline herpesvirus 1, caprine herpesvirus 1, cervid herpesviruses 1 and 2 and elk herpesvirus 1. These viruses share common antigenic properties and the serological relationships between them can be considered as a threat to BoHV-1 eradication programmes. BoHV-1 is a herpesvirus responsible for infectious bovine rhinotracheitis, which is a disease of major economic concern. In this article, the genetic properties of these ruminant alphaherpesviruses are reviewed on a comparative basis and the issue of interspecific recombination is assessed. The pathogenesis of these infections is described with emphasis on the host range and crossing of the host species barrier. Indeed, the non bovine ruminant species susceptible to these ruminant alphaherpesviruses may be potential BoHV-1 reservoirs. The differential diagnosis of these related infections is also discussed. In addition, available epidemiological data are used to assess the potential of cross-infection in ruminant populations. A better knowledge of these ruminant alphaherpesvirus infections is essential to successfully control infectious bovine rhinotracheitis.     

Failure to detect antibody to bovine respiratory syncytial virus in bovine fetal serum.

Sera obtained from 147 bovine fetuses estimated to be between 120 and 270 days of gestation at an abattoir were tested for antibody to bovine respiratory syncytial virus. Antibody to bovine respiratory syncytial virus was not detected in any of the sera examined. Based on the results of this study and a review of the literature, it appears that transplacental infection by bovine respiratory syncytial virus does not occur, or is uncommon.     

DNA restriction enzyme analysis of a bovine herpesvirus 1 strain isolated from encephalitis in Hungary

The DNA of a bovine herpesvirus 1 (BHV-1) strain isolated from calf encephalitis in Hungary was analysed with restriction enzymes. The cleavage pattern of the encephalitis strain Na/67 differed from those of all the other Hungarian BHV-1 isolates investigated so far. The EcoRI and HindIII cleavage patterns of virus strain Na/67 were found to be similar to the patterns of two other encephalitis strains (N569 and A663 from Australia and Argentina, respectively) characterized earlier. Strain Na/67 is the first isolate in Europe which showed the restriction enzyme pattern of BHV-1.3 previously supposed to be characteristic of encephalitis strains.     

Defective infection of bovine herpesvirus 1 in non-permissive murine cells.

The defective growth of bovine herpesvirus I (BHV-1) was analyzed in non-permissive murine embryo fibroblast, BALB/3T3 A31-1-1 (A31) cells. BHV-1 was able to attach and penetrate into A31 cells at similar levels that were seen in semi-permissive cells. Once penetrated into A31 cells, BHV-1 was efficiently transported to nuclei, but the onset of expression of immediate early (IE) protein and viral DNA replication was not observed. These data suggest that the viral replication of BHV-1 in A31 cells is arrested at the point prior to the expression of IE proteins.     

Influence of isoprinosine on bovine herpesvirus type-1 infection in cattle

A study was conducted to determine the in vivo efficacy of isoprinosine (ISO) in calves infected with bovine herpesvirus type-1 (BHV-1). Calves were infected with BHV-1 on day 0 and received ISO daily for 14 days. Clinical signs of disease, shedding of BHV-1, lymphocyte proliferative responses to mitogens, interleukin-2 production, and alveolar macrophage bactericidal activity were monitored during the study. Rectal temperatures were increased (P less than 0.05) in BHV-1 and ISO-BHV-1 calves at days 3 to 7 postinfection (PI). Isoprinosine did not influence BHV-1 shedding in calves. Lymphocyte proliferative responses to phytohemagglutinin (PHA) were lower (P less than 0.01) in BHV-1 calves when compared to control or ISO calves at day 4 PI, but ISO did not ameliorate this effect. Interleukin-2 activity was greater (P less than 0.05) in ISO-BHV-1 calves on days 4 and 8 PI in PHA-stimulated lymphocytes and on day 8 PI in concanavalin A-stimulated lymphocytes when compared to control, ISO or BHV-1 calves. Isoprinosine treatment of BHV-1-infected calves tended to decrease alveolar macrophage bactericidal activity. These data suggest that ISO does not reverse BHV-1 suppression of lymphocyte proliferation, but may enhance IL-2 production in BHV-1 infected calves.