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A total of 6195 cattle were enrolled in this observational study. Serum antibody concentrations to bovine herpesvirus 1 (BHV1) and bovine viral diarrhoea virus (BVDV) were measured at entry to and exit from backgrounding facilities to assess their statuses on arrival and the extent of seroconversion to these viruses during backgrounding. The backgrounding facilities were contiguous with five feedlots in: Queensland (two sites), New South Wales, South Australia and Western Australia. Cattle were held in the backgrounding facilities for a minimum of 29 days and a median of 34 days. On backgrounding facility entry, 32.7% of the study population was seronegative to BVDV, but 85.7% was seronegative to BHV1. After commingling in the backgrounding facilities, of the cattle that were seronegative on backgrounding facility entry, 33.9% and 30.3% showed a serological increase to BVDV and BHV1, respectively. At backgrounding facility exit, when cattle were placed in their feedlots, 19.6% and 59.1% were seronegative to BVDV and BHV1, respectively, and 0.26% were persistently infected with BVDV. There was a strong association between seroincrease to BVDV and seroincrease to BHV1 (P = 0.005) at animal level in cohorts known to contain an animal persistently infected with BVDV.

Background Biosecurity is a key measure to reduce and prevent the introduction of diseases to farms and minimise spread of diseases within a herd. The aim of the study was to characterise the current application of biosecurity measures on dairy cattle farms in Spain along with their bovine viral diarrhoea and infectious bovine rhinotracheitis status. Methods Data on biosecurity measures for 124 dairy herds were collected using a questionnaire. The sanitary status of these farms for bovine viral diarrhoea and infectious bovine rhinotracheitis was also assessed using antibody ELISA. Data were analysed using multiple correspondence analysis and a two-step cluster analysis. Results Three main clusters of farms were identified: clusters 1 and 2 included herds of small and intermediate sizes. These, particularly cluster 1, showed the most deficiencies in the control of vehicles and visitors. However, laboratory tests were always performed on purchased animals. Cluster 3 had the largest herd sizes, with somewhat better biosecurity control of vehicles and visitors. However, farms in this cluster also purchased the most animals, sometimes without testing, and hired external workers more often. Conclusion The study indicated that, in the study population, there are serious shortcomings in the application of biosecurity measures on dairy farms, exposing them to disease transmission. This survey also highlights regional and herd size-related differences in the implementation of biosecurity. Collecting data is an important first step to identification of specific weaknesses in different farm typologies, and an adequate follow-up is needed to ensure that measures are implemented correctly on farms.

Infectious bovine rhinotracheitis (IBR) is the major disease caused by bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle. Being an alphaherpesvirus, the virus establishes a lifelong latent infection in sensory nerve ganglia following primary infection. Latent virus can be reactivated by various stressful stimuli, and subsequent re-excretion of the infectious virus maintains infection within a herd. BoHV-1 is important to all cattle farmers because of the range of diseases it can cause and because infection can be a barrier to international trade; therefore, every cattle herd health plan should contain a strategy for its control. A feature of infections caused by the virus is their variability – from severe and fatal to mild and even subclinical – and outcomes are dependent on combinations of viral, host and environmental factors. An accurate diagnosis is crucial and control of disease is feasible given the excellent diagnostic tools, understanding of the biology of the virus and the availability of effective IBR marker vaccines. Eradication of the virus is also possible over a longer period of time. This article describes the virus and the diseases it causes, and then discusses options for diagnosis, vaccination, control and eradication, and possible priorities for further study.

Contents Infection with bovine herpesvirus-1 (BHV-1), also called infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus, is associated with a variety of respiratory, neurological and infertility health problems causing worldwide economic losses and trading restrictions to the livestock industry. Although there is a considerable amount of information about the risk of BHV-1 transmission through contaminated semen used for artificial insemination, there is no available evidence to indicate whether the resulting embryos, when used for embryo transfer (ET), can lead to the transmission of BHV-1 to recipients and offspring. For this study, cryopreserved bull semen contaminated with BHV-1 was used for artificial insemination (AI) of seronegative, superovulated heifers (N = 43). Embryos were collected post-mortem at 7 days post-insemination and were washed according to the International Embryo Transfer Society (IETS) guidelines. BHV-1 was detected in all samples of follicular fluid, oviductal epithelial cells, endometrium and corpora lutea tissues and a proportion of unwashed (52 of 120, 43%) and washed oocytes and embryos (7 of 113, 6%) collected from embryo donors. Of the 396 collected, unfertilized oocytes and embryos, only 29 (7%) were of ET quality. Most of the embryos and oocytes were degenerated (N = 224, 57%) or unfertilized (N = 143, 36%). The 13 heifers, which each received a single morula-stage washed embryo, maintained seronegative status, but only two (15%) became pregnant and delivered BHV-1-free calves. In conclusion, results suggest that embryos fertilized with BHV-1-contaminated semen may not result in disease transmission to embryo recipients or their offspring when embryos are processed according to IETS and the World Organization for Animal Health (OIE) guidelines. However, due to the transmission of BHV-1 via AI to embryo donors and the apparent adverse effect of BHV-1 on the quality of the embryos, it is unlikely that the procedure can be justified for a commercial application.

The objective of this study was to determine whether a DNA vaccine encoding bovine CD154 linked to a truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD-CD154) induces enhanced tgD-specific immune responses in cattle. In vitro characterization demonstrated that tgD and tgD-CD154 both bind to cultured bovine B cells, whereas only tgD-CD154 induces interleukin-4-dependent proliferation, suggesting that tgD-CD154 specifically binds the CD40 receptor and induces receptor signalling. Calves were immunized with plasmid encoding either tgD or tgD-CD154 by intradermal injection with a needle-free device. After two immunizations, tgD-specific immune responses were observed in both vaccinated groups and after challenge with BHV-1 these responses further increased. Animals immunized with plasmid encoding tgD-CD154 had significantly higher tgD-specific serum titres of immunoglobulins G and A but significantly lower numbers of tgD-specific interferon-gamma-secreting cells than animals immunized with plasmid encoding tgD after BHV-1 challenge. This suggests that the expression of an antigen as a chimeric protein with CD154 can qualitatively alter immune responses in cattle. Since we previously showed that plasmid encoding tgD-CD154 induces significantly enhanced secondary tgD-specific antibody responses in sheep, there appear to be interspecies differences in the immune responses induced by tgD-CD154, which suggests that both proteins in the chimeric molecule may influence protein targeting and the induction of an immune response.

In order to determine the ability of bovine herpesvirus type 1 (BHV-1) to suppress apoptosis, we examined the effects of BHV-1 infection on sorbitol-induced apoptosis on Madin–Darby bovine kidney (MDBK) cells. BHV-1 suppresses sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that BHV-1 has one or more anti-apoptotic genes. To elucidate the molecular mechanisms of apoptosis, expression of some genes encoding apoptosis-inhibiting and -promoting factors were analyzed on BHV-1 infected cells during the process of sorbitol-induced apoptosis. Our results revealed that the expression of bcl-2 and bcl-xL decreased after 5 and 3 h p.i., respectively; while bax and procaspase-3 expression increased with respect to control as a function of p.i. times and at 7 h p.i. they were not observed. We further show that the expression of p53 gene was also enhanced, suggesting that this apoptotic mechanism is p53 dependent. From these results, we propose that BHV-1 has one or more genes encoding apoptosis-inhibiting factors which interfere with the involvement of bcl-2 gene family members and apoptotic pathway, depending upon caspase-3, triggered by sorbitol. J. Cell. Biochem. 89: 373–380, 2003. © 2003 Wiley-Liss, Inc.

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.

MILK antibody testing has played a significant role in cattle disease control and eradication programmes in many countries, particularly in mainland Europe and Scandinavia, since the mid-1980s. In Britain, bulk milk antibody testing is an integral part of the statutory control programmes for brucellosis and enzootic bovine leukosis. It is also used by veterinary diagnostic laboratories, and within formal cattle health schemes, for measuring bovine viral diarrhoea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV)/bovine herpesvirus 1 and Leptospira hardjo infections. A bulk tank antibody test for Mycobacterium avium subspecies paratuberculosis (Johne9s disease) was used for a recent prevalence study in Denmark, but proved unreliable. Milk antibody testing has, however, been successfully developed for several other important cattle infections including salmonella, respiratory syncytial virus and coronavirus. In 1997, the VLA introduced bulk milk antibody testing for BVDV, IBRV and L hardjo in its Regional Laboratories. This article discusses the use of these tests, with particular emphasis on practical and cost-effective approaches.

Two hundred and thirty-seven of 2052 cattle which had not been vaccinated against bovine herpesvirus 1 (BHV-1) were seropositive in a glycoprotein B (gB)-blocking ELISA, but seronegative in a glycoprotein E (gE)-blocking ELISA. In order to detect whether they were latently infected with BHV-1, 10 of them were treated with corticosteroids in an attempt to reactivate putatively latent virus. After successive treatments with dexamethasone and prednisolone, no virus excretion was detected and they showed no increase in antibody titres. In contrast, one gE-seropositive animal re-excreted BHV-1 and had a four-fold increase in antibody titre after the corticosteroid treatments. After slaughter, no BHV-1 DNA could be detected with a sensitive PCR in samples of the trigeminal, cervical and sacral ganglia and spinal cords of the gE-seronegative cattle.

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.

In previous experiments, zona pel- lucida (ZP)-intact in vitro-produced (IVP) embryos incu- bated for 1 hr with 10 6.3 TCID50/ml bovine herpes virus-1 (BHV-1), 10 5.3 TCID50/ml cytopathic (CP) bovine viral diarrhea virus (BVDV) or 10 5.3 TCID50/ml noncyto- pathic (NCP) BVDV showed no signs of virus replication or embryonic degeneration. The aims of the present study were to investigate whether a prolonged pres- ence (24 hr or 8 days) of 10 6.3 TCID50/ml BHV-1 or 10 5.3 TCID50/ml BVDV in an in vitro embryo production system affected the rate of cleavage and embryonic development of ZP-intact embryos, and to point out eventual causes of adverse effects. When virus was present in each step of an IVP system, significantly lower rates of cleavage and blasto- cyst formation of virus-exposed embryos were ob- served, in comparison with control embryos (P , 0.01). When embryos were only exposed to virus during the in vitro fertilization (IVF), the rates of cleav- age and blastocyst formation were significantly af- fected. The introduction of BHV-1 or BVDV during in vitro maturation (IVM) or in vitro culture (IVC) resulted only in significantly lower rates of blastocyst (P , 0.01). In all experiments, virus replication was not detected in the embryonic cells. On the other hand, virus replication was clearly demonstrated in oviductal cells in the co-culture system, resulting in a degenera- tion of these cells. In an additional experiment, syn- thetic oviduct fluid (SOF) without somatic cells was used as an alternative culture system. Even when SOF-embryos were exposed to 10 6.3 TCID50/ml BHV-1 or 10 5.3 TCID50/ml CP, and NCP BVDV, the rates of blastocyst formation of the BHV-1-, CP-, and NCP BVDV-exposed embryos were not different from the unexposed control embryos, 23%, 24%, and 24%, respectively, vs. 27%. Taken together, it can be con- cluded that the virus-induced adverse effects on embry- onic development in conventional co-cultures were due to changes in the embryonic environment caused by infection of oviductal cells. Mol. Reprod. Dev. 54:255- 263, 1999. r 1999 Wiley-Liss, Inc.

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.

Bulk tank milk samples from 1070 dairy herds in England and Wales were tested by ELISA for antibodies to bovine virus diarrhoea virus (BVDV). A subset of 341 herds was tested by ELISA for antibodies to bovine herpesvirus 1 (BHV-1), bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCV). None of the herds had less than 40 dairy cows and none had been vaccinated against BVDV. The prevalence of BVDV antibody-positive herds in the national population was estimated at 95 per cent and approximately 65 per cent of the herds had a high level of bulk tank antibody suggestive of recent infection with BVDV. Dairy herds in East Anglia and the south-east of England had a significantly lower risk of being BVDV antibody-positive than herds in the rest of England and Wales. However, these regional differences tended to diminish with increasing herd size. Around 69 per cent of the herds were BHV-1 antibody-positive and all the herds were antibody positive to BRSV and BCV. Comparison with earlier serological surveys revealed that there had been little change in the prevalence and distribution of BVDV antibody-positive herds in England and Wales over the last 20 years, but that there had been an increase in the prevalence of BHV-1 antibody-positive herds.

A bovine herpesvirus 1 (BHV-1) isolate from the semen of a subclinically infected bull was administered to cattle by various routes to assess its virulence. Cattle that were artificially inseminated or inoculated intrapreputially did not develop clinical signs, but did transmit the virus to contact cattle. However, the isolate induced severe signs of rhinotracheitis and vulvovaginitis in cattle that were inoculated by the intravaginal, intranasal or intravenous routes, but did not infect the fetus. The isolate was therefore not of low virulence. Analysis with DNA restriction enzymes could not assign the isolate to either the BHV-1.1 or BHV-1.2 genotype.

Summary The Frutillar 82, F nasal 90, F vaginal 90, Puente Alto 77, VMLB 79, and Bio Bio 92 isolates of bovine herpesvirus 1 (BHV-1) from Chile were analysed by Eco RI, Bam HI and Hind III restriction enzymes (RE). The Los Angeles (LA) strain was used as reference. The results indicated that the fingerprints of F nasal 90 and F vaginal 90 were identical to the three RE used, but they were different from the other isolates. Major differences were found for Puente Alto 77 and VMLB 79 isolates with Eco RI, and for Puente Alto 77 with Hind III compared with the other Chilean isolates. It is concluded that Chilean BHV-1 isolates obtained from diverse lesions and geographic locations are genomically different from each other and from the other strains described in the literature.

Vaccination has been important in controlling a wide variety of viral and bacterial infections of man and animals. Vaccines to herpesvirus infection of cattle are no exception. The present review describes the different types of conventional vaccines that have been used to date and furthermore describes the novel approaches which are presently being implemented to develop more effective vaccines. These include subunit vaccines as well as genetically engineered modified live deletion mutants. Both these novel vaccine approaches appear to be more efficacious than conventional vaccines. Furthermore, these vaccines provide an additional dimension for control and eradication of infection by providing an opportunity to develop companion diagnostic tests to differentiate infected animals from vaccinated animals. This review summarizes these developments as well as present knowledge regarding the important host defence mechanisms required for preventing infection and aiding recovery from infection.

Summary An intradermal (ID) test (a delayed-type hypersensitivity test) was used for the diagnosis of BHV-1 infection in cattle. A threshold value for the test positive results was established based on the analysis of the agreement between the results of ID test and those of virus neutralization (VN 24) assay. Linear regression analysis revealed no correlation between the intensity of the skin reaction (expressed as an increase in skin-fold thickness) and the VN 24 antibody titre. The sensitivity and specificity of the ID test compared to VN 24 one, were 86.6 % and 98.3 % respectively. Of the 487 cattle with an age of over 6 months, 25 (5.1 %) reacted discordantly by ID and VN 24 tests, i.e. 19 seropositive animals expressed little or no skin reaction, whereas 6 seronegative individuals were positive by ID. Neither of the calves, with passively acquired antibodies in contrast to experimentally infected ones, showed distinctive skin reaction.

Summary The effect of the repeated delayed-type hypersensitivity (DTH) testing (an intradermal test — ID) on virus neutralizing (VN) antibody and DTH responses has been studied. Repeated intradermal injection of inactivated BHV-1 antigen elicited neither the virus neutralizing antibody nor positive skin reaction in cattle free from BHV-1 infection. On the other hand, the same manipulation carried out in a herd infected with BHV-1 provoked seroconversion and/or positive DTH reaction in some of the animals without detectable pre-existing VN antibody. Cattle initially positive by VN and intradermal tests showed steady increase in antibody titre, and some of them developed little or no skin reaction following repeated DTH testing.

A subunit vaccine in the form of immunostimulating complex (iscom) was prepared to contain the envelope glycoproteins of bovine herpesvirus type 1 (BHV-1). This iscom preparation was tested in a vaccination experiment on 4-month-old calves seronegative to BHV-1. In this experiment, four groups with three animals per group were used. Two groups were vaccinated with the iscom preparation twice, four weeks apart, one group with 50 micrograms and the other with 100 micrograms per calf. The third group received a commercial inactivated whole-virus vaccine applying the same vaccination program. The fourth group served as control. Two weeks after the second vaccination, all the animals were challenge-infected intranasally with a virulent BHV-1 strain and four days later with a virulent Pasteurella multocida--this in order to mimic hard field conditions. When exposed to challenge infection, all the animals vaccinated with the iscom were fully protected, i.e., no virus could be recovered from their nasal secretions and no clinical symptoms were recorded. In contrast, the animals vaccinated with the commercial vaccine, responded to challenge with moderate fever and loss of appetite, and virus was isolated from the nasal secretions. The animals in the control group developed severe clinical symptoms. In the sera of iscom-vaccinated animals, the virus neutralization titers reached levels of 1/3500 or higher.

Summary Two field trials were undertaken to evaluate specific vaccines and Biological Response Modifiers (B.R.M.) with regard to disease control of beef cattle. Clinical data and laboratory results strongly suggested a genetically determined resistance to transportation stress of some cattle breeds, with possible important consequences on the occurrence of multi-factor diseases. Plenty of calves showed detectable levels of serum interferon and a remarkable activation of at least some lymphocyte populations in the first days after arrival. In general, B.R.M.-treated cattle could better react against environmental pathogens, as evidenced by both clinical and laboratory findings. Several data emerged that even the efficacy of specific vaccines could be largely accounted for by a positive modulation of non-specific immunity; this latter feature can be mainly referred to adjuvants and/or some bacterial antigens.

Summary Seroepizootiological studies on prevalence of bovine herpesvirus 1 (BHV1) and bovine herpesvirus 2 (BHV2) 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. Zusammenfassung 2800 Rinderseren der Jahre 1984—1987 aus den Rinderzuchtgebieten Namibias wurden auf Antikorper gegen BHV1 und BHV2 mittels Elisa untersucht. Vom Nordosten zum Sudwesten des Landes konnten parallel mit abnehmenden Niederschlagsmengen fallende BHV 2-Antikorperpravalenzen nachgewiesen werden. Bei BHV1 war der Anteil Antikorper-positiver Seren in den nordlichen kommunalen Gebieten ebenfalls am hochsten (bis 80%), im kommerziellen Hauptgebiet der Rinderzucht mit den besten Farmbedingungen am niedrigsten (6–26%), aber in den sudlich daran anschliesenden regenarmeren Zonen, wo schlechtere Voraussetzungen fur die Rinderzucht herrschen, wieder hoher (18–32%).

We studied the morphology and morphogenesis of viral envelopes and nucleocapsids of goat poxvirus (GPV) and infectious bovine rhinotracheitis virus (IBRV) by means of the freeze-fracture technique. The GPV at an early development stage was fractured in its middle, and the envelope was shown to be a bilayer of particles. The mature GPV was fractured between two monolayers of the envelope. These facts suggest that there is little lipid and mainly protein particles in the envelope at the early-development stage, then the lipid inserts into the envelope during viral development. We found that there were still many intramembranous protein particles in protoplasmic fracture face (PF) and extracellular fracture face (EF) of the envelope of mature GPV in the cytoplasm, and fewer particles in the envelope of released GPV. In the envelope of mature IBRV, however, there were many more intramembrane protein particles in the PF face than that in the EF face. Spike-like structures could be seen at the outer edge of the IBRV envelope at times. Protein particles were regularly arranged in the plasmic membranes contacting IBRV. This phenomenon seems to be related to IBRV release. The naked cores, empty capsids, and nucleocapsids of IBRV were assembled in the nucleus of infected cells at the same time. The assembled nucleocapsids could be divided into five types according to their different fracturing positions, whose morphology was observed after deep etching. The morphology of the samples prepared with different methods was compared as well.

Contents: Immune electron microscopy, hybridization (Sandwich, Southern-blot and dot-blot hybridization) as well as two cell culture inoculation techniques to detect bovine herpesvirus 1 (BHV-1) in naturally and artificially contamined bovine semen were compared. Immune electron microscopy was not a suitable method because of its low limit of detection. Dot-blot hybridization, as the most sensitive hybridization technique, could detect 150 pg BHV-1 DNA/semen straw (∼ 106TCID50/ 500 μl semen). Best results were obtained by using cell culture techniques. With the first technique embryonic bovine lung cells (EBLC) on microtiterplates were inoculated with an ultracentrifuged pellet of seminal plasma (pelleting method). The second procedure comprised a dilution of 250 μl (1/2 content of a straw) in 6,25 ml cell culture medium which were overlayed on a confluent layer of EBLC in a 25 cm2 flask. After an incubation of 4 hours at 37°C the medium was decanted and replaced by 6,5 ml fresh medium (dilution method). With both tissue culture methods as little as 5 TCID50 of BHV-1 in 500 μl of artificially contaminated semen were detectable. However the dilution method was better suited than the pelleting method because toxic effects to cells were less pronounced. It was even possible to detect BHV-1 in naturally contaminated semen samples where all other methods failed. The DNA of virus isolates from semen showed three different restriction patterns. Inhalt: Vergleich dreier Methoden zum Nachweis von bovinem Herpesvirus Typ 1 (BHV-1) in naturlich und experimentell kontaminiertem Rindersamen Die Immunelektronenmikroskopie, die Hybridisation (Sandwich-, Southern-Blot- und die Dot-Blot Hybridisation) sowie zwei Zellkultur-Inokulationstechniken wurden zum Nachweis von bovinem Herpesvirus Typ-1 (BHV-1) in naturlich und kunstlich kontaminiertem Stierensamen verglichen. Die Immunelektronenmikroskopie erwies sich dabei als zu wenig empfindliche Methode. Die Dot-Blot Hybridisation war die beste der Hybridbationsmethoden. Es lieβen sich damit bis zu 150 pg BHV-1 DNA/Paillette (∼ 106 TCID50/500 μl Samen) nachweisen. Am besten eigneten sich Zellkulturmethoden. Eine erste Methode bestand darin, embyonale bovine Lungenzellen in Microtiterplatten mit einem Ultrazentrifugensediment des Samenplasmas zu inokulieren (pelleting method). In einem zweiten Verfahren wurde 1/2 Inhalt einer Pailette (250 μl) in 6,25 ml Zellkulturmedium verdunnt und damit ein Zellrasen von embyonalen bovinen Lungenzellen uberschichtet. Nach 4 h bei 37°C wurde das Medium abgegossen und durch 6,5 ml neues Medium ersetzt (Verdunnungsmethode). Mit beiden Methoden lieβen sich 5 TCID50 BHV-1 in 500 μl Runstlich kontaminiertem Samen nachweisen. Die Verdunnungsmethode war aber besser geeignet, weil der toxische Effekt auf die Zellen geringgradiger war. Mit der Verdunnungsmethode lieβ sich aus Ejakulaten BHV-1 isolieren, auch in Fallen, in denen alle andern Methoden versagten. Die DNA's von Virusisolaten aus Rindersamen wiesen drei verschiedene Restriktionsmuster auf.

SUMMARY Restriction endonuclease DNA fingerprints of herpesviruses isolated from 3 unrelated epidemics of bovine encephalitis are similar to each other and totally different from bovine herpesvirus 1 (BHVl). Herpesviruses, antigenically related to BHV1, isolated from goats and buffalo have distinct DNA fingerprints. We propose that bovine encephalitis herpesvirus is prototypic of a new bovine herpesvirus type and that alphaherpesviruses from individual ruminant species are species specific.

The role of bovine respiratory syncytial virus, parainfluenza type 3 virus and bovine herpesvirus 1 as disease agents in 28 groups of young cattle on 19 dairy farms which raised their own replacements was investigated. Bovine respiratory syncytial virus infections occurred in 27, parainfluenza type 3 virus infections in all and bovine herpesvirus 1 infections in three of the 28 groups. Some infections were accompanied by clinical signs while others were entirely subclinical. Clinical respiratory disease was observed on 25 occasions in 20 of the groups. Respiratory disease was associated with a bovine respiratory syncytial virus infection on 15 occasions with parainfluenza type 3 virus infection in four cases and with bovine herpesvirus 1 infection in two cases. In four cases there was no association between the respiratory disease and any of the four virus infections. Bovine respiratory syncytial virus infections caused more serious respiratory problems than parainfluenza type 3 virus infections.