Failure of an in vitro lymphoproliferative assay specific for bovine herpes virus type 1 to detect immunised or latently infected animals

The in vitro lymphoproliferative assay specific for bovine herpes virus type 1 (BHV1) was tested for its ability to predict whether an animal was protected against challenge with virulent BHV1 and for its ability to identify animals latently infected with the virus. Three animals that had been in contact with a field strain of the virus, three that had been vaccinated with a modified live-virus vaccine seven weeks previously, six that had been vaccinated in the same way five months previously, and seven control animals that had had no previous contact with the virus were challenged with virulent BHV1. The 12 animals that had had previous contact with BHV1 all resisted the challenge well or fairly well, but six of them did not react positively in the in vitro lymphoproliferative assay. It was concluded that the assay did not give consistent evidence of the immune status of the animals. Four animals that had had previous contact with a field strain of BHV1 were treated with dexamethasone; they excreted BHV1 irrespective of whether they showed a positive response in the in vitro lymphoproliferative assay.     

Comparison of performance of dairy herds that were or were not vaccinated with a bovine herpes virus 1 marker vaccine in 1998

This study analysed the effects of the use of bovine herpesvirus 1 (BHV1) marker vaccine on the performance of dairy cattle. In Spring of 1999, vaccination of 12 herds with the BHV1 marker vaccine resulted in severe animal health problems and mortality. The vaccines used on these farms were all from a batch that appeared to be contaminated with bovine virus diarrhoea virus type 2. This led to a general call to farmers and veterinary practitioners to report side-effects of this vaccine. As a result, more than 7000 farmers reported symptoms. The information was obtained by means of a questionnaire; there was no control group. To determine the effects of the use of the marker vaccine, it was necessary to perform a study based on objectively acquired information. The information collected by the Royal Dutch Cattle Syndicate and the office of Identification and Registration was complied into herd indices on production, udder health, reproduction, and culling. Two groups of dairy farms that had used the BHV1 marker vaccine (attenuated and inactivated vaccine) were compared with farms that were certified BHV1-free. The analyses were performed based on intra-herd comparisons, meaning that per herd each index calculated over a certain period of time after the use of the marker vaccine was compared to a similar period of time prior to the use of the marker vaccine. A total of 144 comparisons were made. Seven comparisons were statistically significant. In two comparisons, the results were in favour of the BHV1-free farms and in five comparisons, the result were in favour of the vaccinated farms. Thus use of the BHV1 marker vaccine could not be proven to affect herd performance. The sensitivity of the tests was very high, so with a high level of probability even very small differences in indices between groups would have been detected.     

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.     

Identification of an introduced bovine herpesvirus type 1 strain in a closed dairy herd by experimental virus reactivation followed by DNA restriction enzyme analysis

In a closed dairy herd in the province of Utrecht in 1995, nine replacement heifers were erroneously intramuscularly vaccinated with Tracherine, a live virus IBR vaccine. More than 18 months later, serology of the herd revealed that a large part of the herd had developed an antibody response towards BHV1 (62 of 87 animals). To investigate whether Tracherine had recirculated on the farm, four BHV1 antibody positive animals, of which two had been vaccinated with Tracherine, were treated with corticosteroids to reactivate latent BHV1. Two virus isolates were obtained and subsequently analysed by resctriction enzyme analysis. Both isolates were identified as BHV1.1 subtypes. One of the isolates was clearly distinct from Tracherine and was most likely a BHV1 field virus. A BHV1 field virus was most likely introduced into the farm even though the herd was closed, the animals had not been in contact with other cattle, and preventive hygienic measures had been implemented. There was no indication that Tracherine had recirculated.     

Comparison of the sensitivity of in vitro and in vivo tests for detection of the presence of a bovine viral diarrhoea virus type 1 strain

Veterinary vaccines are usually tested for the absence of contaminants. However, the quality control does not always imply that vaccines are not contaminated as, for example, illustrated by the bovine herpes virus 1 (BHV1) vaccine used in The Netherlands in 1999 that contained a small amount of bovine viral diarrhoea virus (BVDV1). Thousands of cows were vaccinated with BHV1 vaccine batches, and the question arose as to whether these small amounts of BVDV1, most likely not detected with in vitro tests, could have infected cattle. More in general, the question was whether the outcome of the in vitro tests, i.e. the in vitro infectivity, was indicative for the infectivity for cattle, i.e. the in vivo infectivity. We therefore carried out in vitro experiments to determine the sensitivity of a BVDV1 isolation assay. In addition, we performed two animal experiments, in which we estimated the lowest dose needed to infect calves with BVDV1. We extrapolated the experimental in vitro and in vivo results from a tissue culture infectious dose (TCID50) to a cattle infectious dose (CID50). We observed a partial response in the calves inoculated with this dose: four out of six calves turned out to be infected. In the tissue culture test, all 20 samples tested negative. The response in vivo, however, was not significantly higher than the in vitro response, which implies that no difference in susceptibility was observed between the animal test and the tissue culture test. Based on the results in our experiments, some cattle may have been infected with BVDV1 after the application of the contaminated BHV1 vaccine during the vaccination campaign. The question remains that how many cattle received contaminated vaccine, and became infected with BVDV1.     

Vaccination of calves with contaminated batches of bovine herpes virus 1 vaccines did not result in infection with bovine virus diarrhea virus

The aim of the experiment was to study whether bovine herpesvirus 1 (BHV1) marker vaccine batches known to be contaminated with bovine virus diarrhoea virus (BVDV) type 1 could cause BVD in cattle. For this purpose, four groups of cattle were used. The first group (n = 4 calves, the positive control group), was vaccinated with vaccine from a batch contaminated with BVDV type 2. The second group (n = 4 calves, the negative control group), was vaccinated with vaccine from a batch that was not contaminated with BVDV. The third group (n = 39 calves), was vaccinated with a vaccine from one of four batches contaminated with BVDV type 1 (seronegative experimental group). The fourth group (n = 6 seropositive heifers), was vaccinated with a vaccine from one of three batches known to be contaminated with BVDV type 1. All cattle were vaccinated with an overdose of the BHV1 marker vaccine. At the start of the experiment, all calves except those from group 4 were seronegative for BVDV and BHV1. The calves from group 4 had antibodies against BVDV, were BVDV-free and seronegative to BHV1. After vaccination, the positive control calves became severely ill, had fever for several days, and BVDV was isolated from nasal swabs and white blood cells. In addition, these calves produced antibodies to BVDV and BHV1. No difference in clinical scores of the other groups was seen, nor were BVDV or BVDV-specific antibody responses detected in these calves; however, they did produce antibodies against BHV1. The remainder of each vaccine vial used was examined for the presence of infectious BVDV in cell culture. From none of the vials was BVDV isolated after three subsequent passages. This indicates that BVDV was either absent from the vials or was present in too low an amount to be isolated. Thus vaccination of calves with vaccines from BHV1 marker vaccine batches contaminated with BVDV type 1 did not result in BVDV infections.     

Outbreak of bovine virus diarrhea on Dutch dairy farms induced by a bovine herpesvirus 1 marker vaccine contaminated with bovine virus diarrhea virus type 2

On 23 February 1999, the Dutch Animal Health Service advised all Dutch veterinary practices to postpone vaccination against bovine herpesvirus 1 (BHV1) immediately. The day before severe disease problems were diagnosed on four dairy farms after vaccination with the same batch of BHV1 marker vaccine. Using monoclonal antibodies, bovine virus diarrhoea virus (BVDV) type 2 was found in the vaccine batch. This paper describes an outbreak of BVDV type 2 infection caused by the use of a batch of modified live BHV1 marker vaccine contaminated with BDVD. Sources of information used were reports of farm visits, minutes of meetings, laboratory results, and oral communications from the people involved. The first symptoms of disease were observed on average six days after vaccination. Morbidity was high on 11 of the 12 farms. On five farms more than 70% of the animals became ill, while on one farm no symptoms could be detected. During the first week after vaccination, feed intake and milk production decreased. During the second week, some animals became clinically diseased having nasal discharge, fever, and diarrhoea. At the end of the second week and at the start of the third week, the number of diseased animals increased rapidly, the symptoms became more severe, and some animals died. Mortality varied among herds. Necropsy most often revealed erosions and ulcers of the mucosa of the digestive tract. In addition, degeneration of the liver, hyperaemia of the abomasum, and swollen mesenterial lymph nodes and swollen spleen were found. On 11 of the 12 farms all animals were culled between 32 and 68 days after vaccination after an agreement was reached with the manufacturer of the vaccine. This was the third outbreak of BVD in cattle after administration of a contaminated vaccine in the Netherlands. The possibilities to prevent contamination of a vaccine as a consequence of infection of fetal calf serum with BVDV are discussed. Improvement of controls to prevent contamination before and during vaccine production, and improvement of the monitoring of side-effects is necessary.     

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.     

Diagnosis and prophylaxis of infectious bovine rhinotracheitis: the role of virus latency

Efficient methods of diagnosis and prophylaxis of infectious bovine rhinotracheitis must consider the concept of latency of the etiological agent, infectious bovine rhinotracheitis virus (Bovine herpesvirus 1; BHV 1). The identification of BHV 1 in nasal mucus samples or a rise in specific antibodies have to be cautiously interpreted, because they can signify either a primary infection or a reexcretion of the virus after reactivation. The isolated virus can also either be a vaccine or a virulent strain. Another aspect of BHV 1 infection diagnosis is the detection of latent carriers, which are able to transmit the virus to uninfected animals; delayed hypersensitivity test seems to be a good candidate. The classical methods of prophylaxis protect the animal against the disease, but they should also impede the reexcretion of virulent strains by latent carriers. Since, in several countries, attenuated viruses are used as vaccines, a special emphasis has to be laid on the persistence of these vaccine viruses in a latent form in the bovine population.     

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.     

Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with BHV1 and PI3 viruses

Peripheral blood mononuclear cells (PBMC) from calves infected with bovine herpesvirus type 1 (BHV1) or parainfluenza 3 virus (PI3) were cultured in vitro in the presence of inactivated specific antigen presented on MDBK cells. In the presence of inactivated antigen, PBMC from both BHV1-infected and control calves produced interferon (IFN)-alpha in 24 hour cultures. Altering the culture conditions did not result in the detection of immune-specific IFN produced by mononuclear cells from BHV1-infected calves. However, spontaneous IFN was detected in the absence of antigen in 24 hour cultures from infected animals: this IFN was pH 2 labile and completely neutralised by antiserum to recombinant bovine IFN-gamma. Spontaneous IFN-gamma production was only seen in calves following a second BHV1 inoculation, given four to seven weeks after the primary dose. In contrast PBMC cultures from PI3 virus-infected calves did not produce IFN-gamma spontaneously, but did so in cultures which contained inactivated PI3 antigen. Mononuclear cells from control animals failed to produce either IFN-alpha or -gamma when cultured with inactivated PI3 virus. IFN-gamma was detected in PBMC cultures after the primary infection, with no increase in production occurring following subsequent PI3 virus inoculations. Immunospecific production of IFN-gamma provides a simple method for monitoring cell-mediated immunity in BHV1- and PI3 virus-infected calves and can be used for evaluating the efficacy of vaccines against these viruses.     

Vaccination to protect calves against infectious bovine rhinotracheitis

A commercially available inactivated vaccine against infectious bovine rhinotracheitis (BHV1) was tested to assess its ability to immunise young seronegative calves and protect them against challenge with a virulent strain of BHV1. Calves showed seroconversion after one or two doses of vaccine. A two-dose and three-dose vaccination regimen each afforded calves significant protection against challenge as judged by the development of clinical symptoms. Vaccinated calves were on average 7 to 10 kg heavier than control calves 24 days after challenge, a statistically significant difference. Vaccination had no significant effect on the virus excretion pattern after challenge.     

The effect of human recombinant interleukin-2 on the porcine immune response to a pseudorabies virus subunit vaccine

The effect of human recombinant interleukin-2 (rIL-2) as an immune enhancing agent was evaluated in pigs vaccinated with a pseudorabies virus subunit vaccine (SV). Two groups of three pigs received two 25 micrograms doses of SV given 3 weeks apart. One group received 10(5) kg-1 day-1 of rIL-2 subcutaneously over two 5-day periods beginning on the day of the first and second vaccine inoculation. Six other pigs were immunized with two 5 micrograms doses of SV. Three of these pigs were treated as above with rIL-2. The effect of treatment was evaluated by comparing: the humoral response; the cell-mediated immune (CMI) response as measured by lymphocyte blastogenesis before and after virus challenge; and the weight response and virus excretion pattern after challenge with virulent pseudorabies virus (PRV). The humoral antibody response as detected by the serum virus neutralization (SN) assay and the enzyme linked immunosorbent assay (ELISA) was consistently higher in rIL-2 treated pigs than in non-treated pigs. These differences were significant (P less than 0.05) among high vaccine dose pigs prior to virus challenge when measured by the SN assay and during the anamnestic response period between days 3 and 10 after challenge when measured by both the SN assay and the ELISA. No differences were detected between treatment groups in the weight response, virus excretion pattern or the CMI response. These results suggest that human rIL-2 may have enhanced the immune response of pigs to the subunit vaccine.     

The effect of a high dose bovine herpes virus 1 marker vaccine in pregnant heifers: virological, bacteriological, immunological, and pathological findings

To determine a possible relationship between the compulsory vaccination against bovine herpesvirus 1 (BHV1) and cattle wasting disease, the effects of BHV1 vaccination on heifers were investigated. Twenty heifers in the third trimester of pregnancy were randomly allotted to a vaccine and a control group. The vaccine group was vaccinated twice with a 50-fold dose of BHV1 vaccine and the control group was inoculated with the diluent. The experiment was performed double blind. After vaccination, the cows were examined daily and condition scores were determined weekly. Blood, milk, and faeces samples were collected weekly for virological, bacteriological, and immunological investigation. The heifers were euthanized either 9 or 13 weeks after the first inoculation and pathological, virological, and bacteriological examination was performed. No differences were detected between the vaccine group and the control group. No concurrent infections were detected and there were no indications of immunosuppression after vaccination. No relationship between the BHV1 vaccination and wasting disease in cattle was detected.     

Vaccine-induced T cell-mediated immunity plays a critical role in early protection against pseudorabies virus (suid herpes virus type 1) infection in pigs

The aim of our study was to evaluate the relative importance of antibody and T cell-mediated immunity in protection against pseudorabies virus (suid herpes virus type 1) infection in pigs. We induced different levels of immune responses by using: (1) a modified live vaccine; (2) the same modified live vaccine with an oil-in-water (o/w) adjuvant; (3) an inactivated vaccine; and (4) the same inactivated vaccine with an o/w adjuvant. Subsequently, we challenged pigs with virulent pseudorabies virus (PRV). We demonstrated that best-protected pigs stood out by maintaining strong T cell-mediated immune (CMI) responses after challenge. Of the immune parameters tested, protection against virus shedding was correlated best with the magnitude of the IFN-gamma response of in vitro re-stimulated peripheral blood mononuclear cells (PBMC) with an additional role for PRV-specific IgG2 antibodies. The use of an o/w adjuvant resulted in higher antibody and CMI responses, in particular with an increased frequency of memory T helper blast cells of in vitro re-stimulated PBMC. However, this adjuvant-induced enhancement of the immune response had a limited additional effect on the efficacy of inactivated vaccines. This study suggests a major contribution of the CMI response in early protection against PRV infection and that PRV-induced IFN-gamma responses may serve as a suitable indicator for assessing the immune status of vaccinated pigs.     

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.     

Reactivation of Bovid herpesvirus 1 and 2 and Parainfluenza-3 virus in calves latently infected

Two experiments were carried out to determine whether Bovid herpsvirus (BHV) 2 is able to induce a recurrent infection in experimentally infected calves. In the first experiment the stress induced by dexamethasone (DMS) treatment failed to reactivate the clinical condition or to induce shedding of BHV2. However, treatment with DMS reactivated a latent BHV1 infection in all calves previously inoculated with BHV2 and also in two noninoculated controls. Probably, because of the interference by BHV1 the study failed to resolve the question as to whether BHV2 could induce a recurrent infection. Consequently, a second experiment was performed using calves devoid of antibody to BHV1 and, therefore, probably, free of virus. By this study it was demonstrated that BHV2 can remain as a latent infection in cattle, which, when immunosuppressed as with DMS, can be reactivated. A finding of considerable interest in this experiment was that in 1 calf a concurrent piroplasma infection was also, unexpectedly, discovered.Recrudescence of latent BHV1 infection was induced by DMS treatment of calves possessing antibody to the virus. The infection once reactivated, was readly transmitted by contact to three other calves devoid of antibody to BHV1. In the same experiment Parainfluenza-3 (PI-3) virus was unexpectedly isolated from all calves. It was speculated that all calves were latently infected with PI-3 virus with concurrent infection by BHV1 acting as a stress inducing PI-3 reactivation.These studies seem to indicate that mixed infections could have an important role in the mechanism involved in the establishment of latent infections and viral reactivation.     

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.     

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.