Advances in BHV1 (IBR) research.

Bovine Herpesvirus Type 1 (BHV1) is the aetiological agent of a number of diseases and not only of IBR, namely infectious pustular vulvovaginitis (IPV), infectious balanoposthitis (IBP), conjunctivitis, encephalomyelitis, mastitis, abortion, enteritis, and lesions in the interdigital space. The serological identical strains differ, however, in some aspects. Typical genital strains usually cause a mild illness, sometimes not even detected clinically, but serologically. They hamper eradication programmes and do not cause IBR when inoculated intranasally. The other--modern--strains are, however, always able to induce a severe disease in the genital tracts. But infection of field or vaccine virus leads to the development of humoral and cell-mediated immunity. The latter is, however, not transmitted to neonates via colostrum. BHV1 antibodies can be found in bovines in all continents, and in many wild species. Prevalences vary greatly depending on herd size and management. Because seronegative cattle play a role in international trade a number of European countries have eradicated BHV1, with very high costs involved. Marker and conventional vaccines can prevent disease but not infection followed by the state of latency. The genomes of several strains, including the marker strains can remain latent in the same animal and be reactivated after stress or injection of corticosteroids. For the detection of humoral antibodies the ELISA is widely used. It is useful for testing bulk milk samples for antibodies derived from field virus and conventional vaccines but not from gE-deleted marker vaccines. Importing countries should consider only vaccinated animals for import. They should require that the animals are seronegative prior to vaccination.     

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.     

Improvement of serological discrimination between herpesvirus-infected animals and animals vaccinated with marker vaccines

Control/eradication plans of bovine herpesvirus 1 (BHV1) and suid herpesvirus 1 (SHV1) infections involve vaccination with inactivated or attenuated gE-deleted marker vaccines and associated companion serological tests to discriminate naturally infected from vaccinated animals. Blocking or competitive enzyme-linked immunosorbent assays (ELISAs) have been designed for the detection of specific antibodies against BHV1 or SHV1 gE glycoprotein. The antigen source usually consists of a crude viral preparation in which gE is associated with other envelope glycoproteins. Such assays suffer from a lack of specificity which is not due to serological cross-reactions with other pathogens. Interestingly, false-positive results occur with sera collected from multivaccinated cattle or pigs. After multivaccination with a marker vaccine, the binding of the conjugated monoclonal antibody used as a tracer, could be hampered by antibodies directed against the other viral glycoproteins.In order to validate the steric hindrance hypothesis, a simple preadsorption of such samples was carried out with a preparation of antigen devoid of gE, prior to the blocking ELISA itself. The decrease in antibody concentrations against the major glycoproteins, clearly leads to a better discrimination between positive and negative samples; that is between infected and multivaccinated animals, without significant loss of sensitivity. This experiment confirms the steric hindrance hypothesis, therefore serum preadsorption could be an easy way to improve the specificity of currently available diagnostic tests.     

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.     

The serological BHV1 status of dams determines the precolostral status of their calves

Precolostral calves and their dams were serologically investigated for the presence of antibodies against Bovine Herpesvirus 1 in diagnostic tests with a very high sensitivity and specificity. Although the syndesmo-chorial type of placenta of ruminants does not transfer gamma globulins, a large number of calves had antibodies, in most cases in a very low concentration. Significant correlations were found between the serological status of the dam, the status of the calf, and the titre of antibodies. Oral intake of maternal blood by the calf at birth or transmission or leakage of maternal antibodies during pregnancy might be possible causes of precolostrally positive calves. From the results it is concluded that to reduce the risk of obtaining BHV1-positive calves, BHV1-negative dams should be selected for breeding purposes.     

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.     

BHV-1 eradication program in Bavaria: a marker-independent strategy

In Bavaria a BHV-1 eradication program was initiated in 1986 and was changed to a compulsory program in 1998. The eradication success increased progressively from < 50% in 1986 to 87% of the farms in 2002. BHV 1-free farms are controlled by bulk milk serology twice a year along with blood serology in animals that are negative but from herds where positive field virus infected animals are present. All serological tests are performed with an indirect ELISA test, all positive results are confirmed by a gB ELISA. Currently about 100.000 virus infected cattle are in Bavarian herds, approximately 80% of these animals are in heavily infected herds (> 10 infected animals). These herds comprise about 5% of all Bavarian herds. The eradication of the virus in these heavily infected herds is the most diifficult, whereas the prevention of new infections appears controllable. In this review current problems in BHV1 eradication are named and possible improvements are discussed.     

The detection of bovine herpesvirus type 1 (BHV 1) using an intradermal test. I. Field studies

An intradermal test (delayed hypersensitivity test) for the diagnosis of BHV1 infection was evaluated in 791 cattle of 16 dairy farms. The skin reactions were compared with the results of serological examinations using a commercial BHV1 ELISA kit (Trachitest). As antigen concentrated, purified and inactivated BHV1 was used. The skin reaction (increase of the skin fold thickness) was used for the interpretation of test results. The best results were obtained with the control of the skin reaction on the third day after injection of the antigen. From 393 serologically BHV1 negative cattle with an age of more than 6 months 391 (99.5%) had a skin reaction up to 1.0 mm and 2 animals (0.5%) had a reaction of 1.3 and 1.9 mm, respectively. The mean increase of skin fold thickness was 0.2 mm. Out of 291 serologically BHV1 positive cattle with an age of more than 6 months 270 had antibodies from natural infection and, partially, from additional vaccination with inactivated BHV1 vaccine. 266 (98.5%) of these animals showed a skin reaction of more than 2.0 mm, in 3 animals (1.1%) a skin reaction up to 1.0 mm was observed and 1 animal (0.4%) had a reaction of 2.0 mm. The mean increase of the skin fold thickness was 6.3 mm. 21 animals had BHV1 antibodies only because of vaccination with inactivated BHV1 vaccine. Only 4 animals had a skin reaction of more than 2.0 mm. Among 107 animals with an age up to 6 months 30 were serologically BHV1 positive and 77 were BHV1 negative. In all animals the skin reaction was less than 1.0 mm, the mean was 0.2 mm.     

BHV1 infections: relevance and spread in Europe.

Infections caused by BHV1 are very common in Europe, but the disease pattern is quite different: the diseases of the genital tract are most common, those of the respiratory tract vary in intensity and prevalence. Digestive disorders connected with BHV1 are in general only observed in calves and mainly in Belgium. Virus strains causing abortion or encephalitis are only present in a few countries. The same is true for BHV1 induced mastitis. Dermatitis and lesions in the interdigital space seem to be a rare event. BHV1 infections are frequently complicated by bacterial secondary infections, but there is evidence that BHV1 infections can occur simultaneously with bovine virus diarrhoea (BVD) and/or parainfluenza-3 (PI 3) virus. The biggest problem associated with BHV1 infection is the ability of the agent to become latent following a primary infection. The genome of the virus probably remains during the life of the animal in the ganglia of the region where the primary infection occurred. No vaccination can overcome this latent stage. By prophylactic vaccination it is possible to prevent an outbreak of clinical disease but it is impossible to prevent infection followed by the establishment of latency. Eradication programmes in Austria, Denmark and Switzerland have removed most of the seropositive cattle from the bovine populations. Currently a sanitary programme is also being conducted in Germany.     

Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4)

A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submitted for diagnostic purposes a good agreement (kappa = 0.75, confidence limits = 0.63-0.88) was found between VN test and ELISA regarding a significant increase in titres. Also, a good correlation was found between VN and ELISA titres (r = 0.76, p<0.0005). The relative sensitivity and specificity of the Mab blocking ELISA as compared with the VN test were 99.9 and 71%, respectively. The rather low relative specificity of the ELISA may be explained by a relatively low sensitivity of the VN test. The ELISA also detected increases in titre after vaccination with an EHV1 subunit vaccine, and after primary field infections in weaned foals. We concluded that the Mab blocking ELISA is more sensitive, easier to perform, more rapid and more reproducible than the VN test. We consider this test as a valuable tool for serological diagnosis of both EHV1 and EHV4 infections.     

Induction of humoral antibodies by a BHV-1 intradermal test]

An intradermal test for the diagnosis of BHV 1 (Intrakutantest Behringwerke AG) was applied to 53 nonvaccinated BHV 1-seronegative cattle aged 7 months to 8 years. Serologic blood testing performed subsequently using ELISA (Enzygnost-IBR/IPV, Behringwerke AG) and SNT revealed seroconversion in 24 of 45 animals without previous maternal antibodies. A second application of the intradermal test after these BHV 1-antibodies had declined, lead to a 'booster-effect' while a control group remained negative. Six of nine animals not affected by the first intradermal application of BHV 1-antigen, developed BHV 1-antibodies following the second intradermal test. Eight animals possessing maternal antibodies showed no serological response to the intradermal test at all. Present results strongly suggest an induction of humoral BHV 1-antibodies by the intradermal application of inactivated BHV 1. Consequently, the indirect control of BHV 1 infections, especially in cattle breeding farms, should be done exclusively by serological examination.     

Risk factors for introduction of BHV1 into BHV1-free Dutch dairy farms: a case-control study

In May 1998, a compulsory eradication programme for BHV1 started in the Netherlands. In December 1999 approximately 24% of Dutch dairy farms were certified BHV1-free (Animal Health Service (AHS)). Ninety-three certified BHV1-free dairy farms participated in a cohort study that investigated the probability of introduction of infectious diseases. The probability of introduction of BHV1 was determined from March 1997 until April 1999. Ninety of these farms remained BHV1-free and could be used as control farms. From January 1997 until March 1998, BHV1 was introduced into 41 BHV1-free dairy farms in the Netherlands (case farms). Management data were collected for both cases and controls and were complete for 37 case farms and 82 control farms. For small data sets and for data in which both low and high frequencies were expected in the contingency tables, the asymptotic methods were unreliable. Our data set clearly resembled such a data set; the risk factors were rare events because the BHV1-free farms were closed farms on which few direct animal contacts occurred. Therefore, an exact stratified modelling approach was most suitable for the data. The study showed that dairy farms should prevent cattle from escaping or mingling with other cattle and that professional visitors should always wear protective farm clothing.     

Airborne transmission of BHV1, BRSV, and BVDV among cattle is possible under experimental conditions

To control the diseases caused by bovine herpesvirus 1 (BHV1), bovine respiratory syncytial virus (BRSV), and bovine virus diarrhoea virus (BVDV), it is crucial to know their modes of transmission. The purpose of this study was to determine whether these viruses can be transmitted by air to a substantial extent. Calves were housed in two separate isolation stables in which a unidirectional airflow was maintained through a tube in the wall. In one stable, three of the five calves were experimentally infected with BHV1 and later with BRSV. In the BVDV experiment, two calves persistently infected with BVDV (PI-calves) instead of experimentally infected calves, were used as the source of the virus. In all the calves infections were monitored using virus and antibody detection. Results showed that all the three viruses were transmitted by air. BHV1 spread to sentinel calves in the adjacent stable within three days, and BRSV within nine days, and BVDV spread to sentinel calves probably within one week. Although airborne transmission is possibly not the main route of transmission, these findings will have consequences for disease prevention and regulations in control programmes.     

Simulated hazards of loosing infection-free status in a Dutch BHV1 model

A compulsory eradication programme for bovine herpesvirus 1 (BHV1) was implemented in the Netherlands in 1998. At the start of the programme, about 25% of the dairy herds were certified BHV1-free. Simulation models have played an important role in the decision-making process associated with BHV1 eradication. Our objective in this study was to improve understanding of model behaviour (as part of internal validation) regarding loss by herds of the BHV1-free certificate. Using a Cox proportional hazards model, the association between farm characteristics and the risk of certificate loss during simulation was quantified. The overall fraction of herds experiencing certificate loss amongst initially certified during simulation was 3.0% in 6.5 years. Factors that increased risk for earlier certificate loss in the final multivariable Cox model were higher 'yearly number of cattle purchased', 'farm density within a 1 km radius' and 'cattle density within a 1 km radius'. Qualitative behaviour of risk factors we found agreed with observations in field studies.     

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.     

A comparative study of serological tests for use in the bovine Herpesvirus 1 eradication programme in The Netherlands

We compared a gB-ELISA, a gE-ELISA and a Danish test system (consisting of a blocking and an indirect ELISA) for their specificity and sensitivity to detect antibodies against BHV1. The Danish test system showed the highest sensitivity and the gE-ELISA the lowest; the gB-ELISA showed an intermediate sensitivity. If the doubtful zone (25–50% blocking) of the gB-ELISA was considered as positive (gB-ELISA⁺), the sensitivity almost reached that of the Danish test system. The specificity of all tests appeared to be very high, 99.7, 96.7 100, 99.7% for the gB-ELISA, gB-ELISA⁺, gE-ELISA and the Danish test system, respectively. Seroconversion was detected in the gE-ELISA up to 3 weeks later than in the gB-ELISA and the Danish test system. It is concluded that the combination of a gB-ELISA (for screening) and the Danish test (for confirmation) system used in the BHV1 eradication programme in the Netherlands, provides for very high sensitivity (>99.0%) (Kramps et al., 1994) and a very high specificity (>99.9%).     

Seroepizootiologic studies of the distribution of bovine herpesvirus type 1 and 2 (BHV 1, BHV 2) in Namibia

From cattle breeding areas of Namibia a total of 2,800 sera from 1984-1987 were tested by Elisa for antibodies to BHV1 and BHV2. From north-east to south-west a drop in BHV2 antibody prevalences was found which paralleled the decrease in rainfall. With BHV1 the percentage of positive reacting sera was also highest in the northern communal areas (up to 80%). In the commercial farming area with best farming conditions the percentage was lowest, but it was south of here again higher in the areas of lower precipitation where farming conditions are less optimal.     

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.     

Sheep do not have a major role in bovine herpesvirus 1 transmission

With regard to BHV1 eradication programs in cattle it is important to know whether sheep can be a reservoir of BHV1. We therefore performed an experiment that consisted of three phases. In phase 1, 10 sheep were inoculated with high doses of BHV1 and kept in close contact with 5 sheep and 5 calves. All inoculated sheep excreted BHV1 between 8 and 15 days post inoculation and seroconverted. Although BHV1 was isolated from the nasal mucosa of 3 out of 5 sentinel sheep, none of the sentinel sheep produced antibodies against BHV1. One sentinel calf excreted BHV1 through days 12–17; the remaining 4 calves excreted BHV1 between days 18 and 24, suggesting that the first calf was infected by sheep and the remaining 4 sentinel calves were infected by that calf and not by sheep. The bacic reproduction ratio (R₀) of BHV1 between sheep and calves was estimated at 0.1, and among calves it was estimated at ≥9. In phase 2, all inoculated sheep were treated with dexamethasone and kept in close contact with 5 sheep and 5 calves. All dexamethasone treated sheep re-excreted BHV1 over a 6- to 9-day period. None of the sentinel animals seroconverted. In phase 3, the sentinel sheep and calves of phase 1 were kept in two groups and were treated with dexamethasone. None of the sentinel sheep re-excreted BHV1, whereas 3 out of 5 sentinel calves did. It is concluded that while BHV1 infection in sheep is possible, BHV1 does not spread from sheep easily to cattle.     

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.