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.     

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.     

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.     

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.     

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.     

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.     

Determination of BHV1 specific immune reactivity in naturally infected and vaccinated animals by lymphocyte proliferation assays

The in vitro BHV1-specific lymphocyte stimulation assay was used to investigate immune reactivity of cattle after natural infection or vaccination with BHV1. Proliferative responses to live virus were shown in tests with peripheral blood lymphocytes of seropositive field virus-infected animals and of vaccinated animals. Nineteen out of 36 seropositive field virus-infected animals did not show in vitro responses. Nine out of 12 animals showed, at least transient, responsiveness after vaccination. Antibody titers were maintained throughout the observation period. T cell activity is believed to play a role in protection against BHV1 infection. The in vitro proliferative assay, however, can not discriminate between BHV1 seropositive and seronegative field virus-infected animals. After vaccination, the BHV1-specific lymphocyte responses of at least one animal disappeared. Both observations may point to the fact that T cell memory is generated, or at least systemically present, to a limited extent.     

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

A purified concentrated BHV1 antigen that had been tested in field trials was also tested under isolated conditions in BHV1 positive and negative cattle. The antigen proved to act specifically even after storage for 2 years at +4 degrees C or for 6 months at 37 degrees C. The best results were obtained when the test was read 48-72 hours after the injection, which is in agreement with the field trials. The increase in skin thickness decreased unless boosted by infection or vaccination gradually. The test is unsuitable to control a vaccination programme. There was no correlation between the increase in thickness and humoral antibody titers. Repeated application of the intradermal injection of the antigen did not result in seroconversion in seronegative cattle. The biological limits of the test evaluation are discussed.     

Evaluating control strategies for outbreaks in BHV1-free areas using stochastic and spatial simulation

Several countries within the EU have successfully eradicated bovine herpesvirus type I (BHV1), while others are still making efforts to eradicate the virus. Reintroduction of the virus into BHV1-free areas can lead to major outbreaks - thereby causing severe economic losses. To give decision-makers more insight into the risk and economic consequences of BHV1 reintroduction and into the effectiveness of various control strategies, we developed the simulation model InterIBR. InterIBR is a dynamic model that takes into account risk and uncertainty and the geographic location of individual farms. Simulation of a BHV1-outbreak in the Netherlands starts with introduction of the virus on a predefined farm type, after which both within-farm and between-farm transmission are simulated. Monitoring and control measures are implemented to simulate detection of the infection and subsequent control. Economic consequences included in this study are related to losses due to infection and costs of control. In the simulated basic control strategy, dairy farms are monitored by monthly bulk-milk tests and miscellaneous farms are monitored by half-yearly serological tests. After detection, movement-control measures apply, animal contacts are traced and neighbour farms are put on surveillance. Given current assumptions on transmission dynamics, we conclude that a strategy with either rapid removal or vaccination of infected cattle does not reduce the number of infected farms compared to this basic strategy - but will cost more to control. Farm type with first introduction of BHV1 has a considerable impact on the expected number of secondarily infected farms and total costs. To limit the number of infected farms and total costs due to outbreaks, we suggest intensifying the monitoring program on farms with a high frequency of cattle trade, and monthly bulk-milk testing on dairy farms.     

Adaptive conjoint analysis to determine perceived risk factors of farmers, veterinarians and AI technicians for introduction of BHV1 to dairy farms

A study was carried out to determine the possibility of a more-closed farming system for (Dutch) dairy farms. The objective of the study was to provide effective and economically profitable management advice for improving the animal-health status of farms. Management measures will only be successfully applied if supported by farmers and their advisors (such as veterinarians). Therefore, the perception of farmers and advisors of the importance of various risk factors for the introduction of diseases to a farm was determined by using bovine herpes virus type 1 (BHV1) as an example.

As part of the study, an evening-long workshop was organized and run thrice. In total, 49 farmers, veterinarians and AI technicians participated in these workshops. The computerized questionnaire technique was based on adaptive conjoint analysis (ACA). ACA has the advantage that participants can work with a large number of risk factors in a relatively short period of time. Another advantage of ACA (compared with standard questionnaires) is that the answers from each participant can be checked with regard to consistency with respect to the importance assigned to them. Data from participants with inconsistent responses can be excluded from further analyses. The results of the ACA interview were compared with the risk factors reported in the literature as being associated with BHV1 status (e.g. purchase of cattle, participation in cattle shows) and with farmers' actual management to prevent the introduction of diseases.

The workshop participants were all operating in the dairy sector and they seemed well aware of the risk of direct animal contacts for the introduction of BHV1. Farmers thought visitors to be more risky than did AI technicians and (especially) veterinarians. Farmers who purchased cattle or participated in cattle shows were of the opinion that the risks of direct animal contacts were more important than did farmers who were not involved in those practices. Farmers whose farms were BHV1-positive (and participated in cattle shows more often) thought the risk of participation smaller than did farmers with BHV1-negative farms.     

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.     

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.     

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%     

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.     

Isolation of a herpesvirus serologically related to bovine herpesvirus 1 from a reindeer (Rangifer tarandus)

Serological evidence of exposure of reindeer (Rangifer tarandus) to a virus related to bovine herpesvirus 1 (BHV1) (Synonym: Infectious bovine rhinotracheitis (IBR) virus) has been reported in Canada (El Azhary 1979) and the USA (Dieterich 1981). A serological survey conducted in Finnish Lapland also detected neutralising antibodies to BHV1 in reindeer sera; 23 % of 300 reindeer had detectable antibodies, whereas none of 300 cattle sera from the same region contained antibodies to BHV1 (Ek-Kommonen et al. 1982). There is currently no evidence of BHV1 infection of cattle in Finland, so the isolation and characterisation of the reindeer herpesvirus was of considerable interest. This short communication describes the isolation and preliminary characterisation of a herpesvirus from a reindeer following the administration of dexamethasone.     

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.     

BOVINE HERPESVIRUS 1 - INFECTION OF THE UPPER ALIMENTARY TRACT OF CATTLE AND ITS ASSOCIATION WITH A SEVERE MORTALITY

SUMMARY A severe cattle mortality in which 132 out of 340 animals died on a property in southern Queensland was investigated. Clinical signs shown by affected animals included fever, inappetance, depression, lethargy, salivation, diarrhoea, ataxia, and ulceration of the oral cavity. The most common lesions seen at autopsy of 6 affected animals were ulceration of the tongue, gums, dental pad and buccal mucosa, linear ulceration of the caudal third of the oesophagus, mild catarrhal enteritis and necrosis of lymph nodes draining areas of ulceration. Bovine herpesvirus type 1 (BHV 1) was isolated from 3 out of 5 animals from which virus isolation was attempted. BHV 1 was recovered from oesophageal ulcers, retropharyngeal lymph nodes, blood clot, and swabs from ulcers in the oral cavity but not from spleen, liver or mesenteric lymph node. Serum neutralising (SN) antibody to BHV 1 was detected in 4 out of 12 affected animals in the second of paired serum samples but not in the first. Mucosal Disease (MD) virus was not recovered from any of 17 animals from which isolation was attempted but moderate MD SN titres, without a rise on paired sera, were detected in affected animals. Fever, depression, inappetance, ulceration of the upper alimentary tract, and adrenal necrosis were produced in 2 susceptible animals following inoculation with third passage cell culture fluid containing BHV 1. A serological response to BHV 1, but not to MD virus was detected in one of the cattle infected experimentally.     

A study in calves of an immunologic relationship between herpes simplex virus and Bovid herpesvirus 2

Calves were inoculated subcutaneously with Herpes simplex virus (HSV), types 1 and 2, previously inactivated with Triton X100. Thirty-nine days later the calves were challenged either by intradermal or intravenous injection of Bovid herpesvirus 2 (BHV2).The clinical reponse of HSV preimmunized calves to BHV2 infection was milder than that in the challenge control calves, and the titer of BHV2 underwent a reduction in the preimmunized calves. BHV2 apparently enhanced the immuno-competent system of the preimmunized calves to produce antibody to HSV.From these results it appeared that HSV partially protected calves against experimentally induced BHV2 infection.     

Cross-reactions of bovine herpesvirus 1 antigens with those of other cattle herpesviruses

Bovine embryonic kidney cells were infected with bovine herpesviruses (BHV1, 2, or 3), suid herpesvirus 1 (SHV1), or were sham-inoculated. When cytopathic effect was apparent, the cells were solubilized using Triton X-100 detergent. Resulting antigen preparations were tested by 2-dimensional immunoelectrophoresis using bovine fetal serum and antisera directed against BHV1, BHV2, BHV3, SHV1 or a restricted spectrum of BHV1 antigens. Interaction of BHV1 antiserum with BHV1 antigen preparations resulted in 11 precipitation arcs. The same antiserum produced 3 arcs with BHV2, none with BHV3, and 5 with SHV1. The interaction of BHV1 antigen preparations with BHV2, BHV3, or SHV1 antisera failed to produce demonstrable arcs. However, when heterologous antigen or antibody preparations were added to BHV1 homologous 2-dimensional immunoelectrophoresis tests, all 11 BVH1 arcs were modified by BHV1, 2 by BHV2, 4 by BHV3 and 4 by SHV1 preparations. Two antigens were common to the 4 herpesviruses. Antigen preparations were tested for their ability to inhibit virus neutralization by BHV1 antiserum; only the BHV1 preparation was active. Sera were tested for BHV1 neutralizing activity; only BHV1 antiserum and a serum specific for a restricted spectrum of BHV1 antigens were active. A glycoprotein antigen associated with BHV1 neutralization was identified which may be important in the protection of animals against disease.