Neutralizing antibodies to bovine herpesvirus 1 in reindeer

Serum samples from cattle and reindeer in Lapland were examined for neutralizing antibodies to the IBR/IPV virus. All the bovine sera tested were negative. The reindeer sera were tested using 2 different virus neutralization methods differing in the serum-virus incubation time prior to inoculation into tissue culture tubes. 12.6 % of the samples tested with a preincubation of 1 h at 37°C were positive, whereas 23 % of those tested with a preincubation time of 24 h at 37°C were positive. The fairly high prevalence of antibodies to IBR/IPV in the reindeer population in Finland indicates the occurrence of the IBR/IPV virus or a closely related cross-reacting herpesvirus.     

An evaluation of five serological tests for the detection of antibody to bovine herpesvirus 1 in vaccinated and experimentally infected cattle

More than 300 bovine sera from a previously reported vaccination and challenge trial were tested for antibodies to bovine herpesvirus 1 (BHV1) by five serological assays: enzyme-linked immunosorbent assay (ELISA) for IgM and IgG, passive haemagglutination (PHA), and two methods of virus neutralisation (VN). In a statistical comparison of ELISA (IgG), PHA and VN results, the assays showed highly significant correlations (P less than 0.01). The sensitivities of ELISA and 24-hour neutralisation tests were similar, in contrast to passive haemagglutination and one hour neutralisation which failed to detect BHV1 antibodies in some low titre sera.     

Airborne transmission of bovine herpesvirus 1 infections in calves under field conditions

A small scale transmission experiment was performed with bovine herpesvirus 1 (BHV1) in a cattle population under field conditions. 10 calves were housed under strict hygienic conditions, with a distance of 4m between each calf. Five calves were experimentally infected with BHV1, two calves with strain Harberink and three with strain Lam, respectively. Experimentally infected calves were placed at 4 m distance from five susceptible sentinel calves. Airborne transmission to sentinel calves was detected using virus isolation and BHV1 specific polymerase chain reactions in samples of nasal fluids, and BHV1 specific antibodies in serum samples. Strain Harberink was hardly transmitted to sentinel calves, whereas strain Lam was transmitted to all sentinels. Estimating the rate of transmission per day, the total number of calves infected by one (strain Lam) infected calf was 1.18. Comparing this estimated transmission ratio between cattle at a distance of 4 m to the estimated transmission ratio R of BHV1 in susceptible commingled cattle reported before, the effect of the factor distance on the transmission ratio could be calculated. Extrapolating these results, a distance of 4.4 m between cattle populations would be necessary to reduce transmission for this strain to R<1.     

Antibodies against bovine herpesvirus (BHV) 5 may be differentiated from antibodies against BHV1 in a BHV1 glycoprotein E blocking ELISA

We examined whether antibodies against bovine herpesvirus (BHV) 5 cross-react with BHV1 antigens and whether they could interfere with BHV1 eradication programmes. Six calves were experimentally infected with different doses of BHV5 strain N569; homologous antibodies were first detectable on day 11 post infection; they cross-reacted in a BHV1 virus neutralisation test, in a BHV1-glycoprotein (g)-B blocking ELISA and in a BHV1-gE ELISA, but not in a BHV1-gE blocking ELISA. This study indicates that, in ongoing BHV1 eradication programmes, based on vaccines that lack gE, BHV5 infections may not lead to false-positive serological reactions in case cattle are tested for BHV1-gE antibodies by the BHV1-gE blocking ELISA; antibodies against BHV5 may be differentiated from antibodies against BHV1. The BHV1-gE blocking ELISA may, therefore, offer opportunities for the serological differentiation between BHV1 and BHV5 infections.     

Evaluation of tests for antibodies against bovine herpesvirus 1 performed in national reference laboratories in Europe

Sets of serum and milk samples were collected from various countries and prepared, lyophilised and distributed by 1 laboratory to 12 reference laboratories in Europe. The serum sets contained the three European bovine herpesvirus 1 (BHV1) reference serum samples (EU1, EU2 and EU3), serum samples from naturally and experimentally BHV1-infected cattle, from vaccinated, and vaccinated-challenged cattle, from uninfected cattle, and a series of serum dilutions. In addition, sets of milk samples were distributed. The samples were tested for antibodies against BHV1 in virus neutralisation tests, in gB-specific ELISAs, in indirect ELISAs and in gE-specific ELISAs. It was found that the virus neutralisation test and the gB-specific ELISAs were most sensitive for the detection of antibodies in serum, whereas for assaying milk samples the indirect ELISAs were the tests of choice. The results show that the quality of most laboratories appeared to be adequate, but that one laboratory performed considerably below an acceptable level of quality. Four samples from the panel have been proposed that might be selected as reference sera in addition to the three European reference samples.     

Aujeszky's disease ELISA: cross-reactions with other herpesvirus antisera

The production of antibodies in pigs to 11 herpesviruses was investigated in relation to their ability to cross-react with Aujeszky's disease virus (suid herpesvirus 1--SHV1). Of the herpesviruses tested only two, sheep herpesvirus (caprine herpesvirus 1) and dog herpesvirus (canid herpesvirus 1), failed to produce homologous virus antibodies. Only the antibodies to bovine herpesvirus 1 (BHV1) produced a cross-reaction by SHV1 enzyme-linked immunosorbent assay (ELISA). No SHV1 neutralizing antibodies were detected in any of the herpesvirus antisera. A cross-reaction with SHV1 by a serum from a pig naturally infected with BHV1 or with any of the other herpesviruses tested was considered unlikely.     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

Bovine herpesvirus 4-associated postpartum metritis in a Spanish dairy herd

In more than 10 Spanish dairy cows, a bovine herpesvirus 4 (BHV4) associated postpartum metritis was confirmed by virus isolation, BHV4-glycoprotein B (gB) PCR and/or serology. In this study, 12 cows with, and, at the time of sampling, 3 cows without clinical signs of acute postpartum metritis from one large dairy herd in Spain were examined for bacterial and viral infections. Blood, placenta/caruncles and uterine contents were collected between day 1 and day 20 post-calving, and examined for the presence of bacteria and for viruses by virus isolation, BHV4 DNA by BHV4-gB PCR and/or BHV4 antibody titres. Bovine herpesvirus 4 was detected in 83% of the cases with clinical signs of acute postpartum metritis by virus isolation and/or BHV4-gB PCR. An increase of BHV4 antibodies was detected in all examined postpartum metritis cows and in the 3 cows without clinical metritis. Two of these 3 cows developed severe metritis a few dayss after collecting the first blood sample. A concurrent infections of BHV4 and bacteria, mainly Arcanobacterium pyogenes and Streptococcus sp., were detected in 73% of the examined uterine contents collected from postpartum metritis affected cows. This case-report study showed a clear association between BHV4 infections and acute postpartum metritis in dairy cows. In addition, the BHV4-associated postpartum metritis appeared to be an emerging syndrome in this Spanish herd.     

Serologic and virologic studies of selected cattle with antibodies to bovine herpesviruses

In order to investigate the specificity of low titer antibodies to BHV 1, twelve cattle were subjected to stress and dexamethasone treatment. They were monitored virologically by inoculating cell cultures with naso-pharyngeal-, ocular- and vaginal- or preputial swabs and serologically by assessing the prevalence and incidence of antibodies to bovine, caprine-, porcine-, and equine herpesviruses and to bovine leukemia virus. Antibodies were classified as specific for BHV 1 if the animals excreted IBR virus, or if the antibodies neutralized BHV 1 and reacted with BHV 1 antigens, or if they reacted additionally with CapHV antigens. Animals whose sera recognized BHV 1 and BHV 2 but not other herpesviruses, were judged to have experienced both infections. Nine of the twelve animals had specific BHV 1 antibodies. With three animals the question for specificity of their antibodies remains open. Two animals experienced several herpesvirus infections. Therefore, the induction of crossreacting antibodies, directed against epitopes common to herpesviruses, could not be ruled out. The sera of one animal reacted with BHV 1 and BHV 4 antigens in ELISA tests. They did, however, not neutralize BHV 1.     

Malignant catarrhal fever: polymerase chain reaction survey for ovine herpesvirus 2 and other persistent herpesvirus and retrovirus infections of dairy cattle and bison.

Using a polymerase chain reaction (PCR) test for sequences of ovine herpesvirus 2 (OHV2), this virus was shown to be significantly associated with sheep-associated malignant catarrhal fever (SA-MCF) in terminal cases of disease in 34 cattle and 53 bison. Ovine herpesvirus 2 was not detected in cattle (38) and bison (10) that succumbed to other diseases. Other persistent herpesviruses, retroviruses, and pestivirus, some of which have been previously isolated from cases of SA-MCF, were not associated with the disease. These included bovine herpesvirus 4 (BHV4), bovine lymphotrophic herpesvirus (BLHV), bovine syncytial virus (BSV, also known as bovine spumavirus), bovine immunodeficiency virus (BIV), and bovine viral diarrhea virus (BVDV). A PCR survey for OHV2 in DNA from individual cow's peripheral blood lymphocytes in 4 dairies showed that the 1 dairy that was in close contact to sheep had a prevalence of OHV2 of 21.3%, whereas the 3 other dairies had no OHV2. Prevalence of the other herpesviruses and retroviruses in the dairy cows was variable, ranging from 2% to 51% for BHV4, 52% to 78.7% for BLHV, and 10% to 34% for BSV. Bovine lymphotrophic herpesvirus and BSV were also found in a few (1-4 of 21 tested) cases of terminal SA-MCF, but BIV and BVDV were not found in either the dairy cows sampled, or in the cases of SA-MCE No significant correlation was found between the presence of any 2 viruses (OHV2, BHV4, BLHV, BSV) in the dairy cows or terminal cases of SA-MCE     

Strains of bovine herpesvirus 1 that do not express an epitope on glycoprotein E in cell culture still induce antibodies that can be detected in a gE-blocking ELISA

Two bovine herpesvirus 1 (BHV1) field strains that do not express an epitope on glycoprotein E (gE) in cell culture were inoculated into calves to examine whether their sera became positive in a gE-blocking ELISA that detects antibodies against gE. This gE-blocking ELISA uses one monoclonal antibody that is directed against the above mentioned epitope. All calves, except one, infected with these gE-epitope negative BHV1 strains, became positive in this gE-blocking ELISA, about two weeks later than in another gE-ELISA and a gB-ELISA. However, cattle infected with BHVI strains that do express this particular gE-epitope showed a similar type of antibody responses. These findings demonstrate that BHV1 strains that do not express a particular gE-epitope in cell culture, still can induce antibodies that are detected in a blocking ELISA that measures antibodies against that epitope.     

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.     

Reactivation of latent bovine herpesvirus 1 in cattle seronegative to glycoproteins gB and gE

Six heifers were vaccinated intranasally with the live bovine herpesvirus 1 (BHV1) temperature-sensitive (ts) vaccine strain RBL106 within 3 weeks of birth. These calves most likely still had maternal antibodies against BHV1. Thereafter, these heifers were vaccinated several times with an experimental BHV1 glycoprotein-D (gD) subunit vaccine. At the age of 3 years these 6 heifers were seronegative in the BHV1 gB and gE blocking ELISAs, but had neutralizing antibodies against BHV1, probably induced by the vaccinations with the gD subunit vaccine. Five of these 6 heifers excreted BHV1 after treatment with dexamethasone. Restriction enzyme analysis of the genome of the excreted viruses revealed that all 5 isolates had a BHV1.1 genotype and that isolates of 3 heifers were not obviously different from the ts-vaccine strain. The restriction enzyme fragment pattern of the isolate of 1 heifer was clearly different from the pattern of the ts-vaccine strain. It is concluded that cattle can be seronegative against BHV1 gB and gE but can still carry BHV1 in a latent form. This finding strongly suggests that there are completely BHV1 seronegative cattle that are latently infected with BHV1. The impact of this finding on BHV1 eradication programmes is discussed.     

Eradication of infectious bovine rhinotracheitis in Switzerland: review and prospects

Ten years after the first outbreak of infectious bovine rhinotracheitis (IBR) in Swiss dairy cows, the national cattle herd is almost free from infection with IBR virus (bovine herpesvirus 1, BHV 1). The national programme for the eradication of IBR was divided into four phases: (1) Prevention of transmission of the infection by restrictions on trade of bovines and assessment of the prevalence of cattle with antibodies to BHV 1. (2) Slaughtering animals with antibodies to BHV 1 in order to eradicate BHV 1 from breeding herds. (3) Detection and eradication of further BHV 1 reservoirs (e.g. fattening cattle). (4) Monitoring programme and legal actions in order to maintain the favourable situation. Approximately 50,000 animals were slaughtered in the course of the eradication of IBR. The total costs amounted to approximately SFr. 110,000,000 over 10 years. The costs for maintaining the situation are estimated at approximately SFr. 5,000,000 per annum.     

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.     

Bovine herpesvirus 1 (BHV1) seroprevalence in the breeding cattle population of the Veneto region: prospects for the implementation of a control programme

The results of a serological survey for bovine herpes virus (BHV1) antibodies in the breeding cattle population of the Veneto region are presented. The data do not support the hypothesis of an high prevalence of BHV1; on farms where vaccination was not carried out most animals were seronegative, and seropositive animals were generally older. Therefore, when drawing up the guidelines for a control programme, systematic immunization (with glycoprotein E-deleted vaccines) should be restricted only to farms with a high prevalence of BHV1 antibodies and/or with a high risk of BHV1 occurrence; in most unvaccinated farms a 'test and removal' policy appears to be more appropriate in order to rapidly eradicate BHV1 from the entire stock.     

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.     

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.     

Protective effects against abortion and fetal infection following exposure to bovine viral diarrhea virus and bovine herpesvirus 1 during pregnancy in beef heifers that received two doses of a multivalent modified-live virus vaccine prior to breeding

Objective-To determine whether administration of 2 doses of a multivalent, modified-live virus vaccine prior to breeding of heifers would provide protection against abortion and fetal infection following exposure of pregnant heifers to cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) and cattle with acute bovine herpesvirus 1 (BHV1) infection. Design-Randomized controlled clinical trial. Animals-33 crossbred beef heifers, 3 steers, 6 bulls, and 25 calves. Procedures-20 of 22 vaccinated and 10 of 11 unvaccinated heifers became pregnant and were commingled with 3 steers PI with BVDV type 1a, 1b, or 2 for 56 days beginning 102 days after the second vaccination (administered 30 days after the first vaccination). Eighty days following removal of BVDV-PI steers, heifers were commingled with 3 bulls with acute BHV1 infection for 14 days. Results-After BVDV exposure, 1 fetus (not evaluated) was aborted by a vaccinated heifer; BVDV was detected in 0 of 19 calves from vaccinated heifers and in all 4 fetuses (aborted after BHV1 exposure) and 6 calves from unvaccinated heifers. Bovine herpesvirus 1 was not detected in any fetus or calf and associated fetal membranes in either treatment group. Vaccinated heifers had longer gestation periods and calves with greater birth weights, weaning weights, average daily gains, and market value at weaning, compared with those for calves born to unvaccinated heifers. Conclusions and Clinical Relevance-Prebreeding administration of a modified-live virus vaccine to heifers resulted in fewer abortions and BVDV-PI offspring and improved growth and increased market value of weaned calves.