Bovine herpesvirus-1 and parainfluenza-3 virus interactions: clinical and immunological response in calves.

Calves infected with bovine herpesvirus-1 (BHV-1) or both BHV-1 and parainfluenza-3 virus (PIV-3) developed clinical signs including fever, cough, and nasal and ocular discharges. Animals infected with both viruses appeared more depressed and showed higher rectal temperature, while calves inoculated with PIV-3 alone had a very mild clinical disease. Both BHV-1 and PIV-3 were recovered from nasal secretions up to six to eight days postinoculation. However, the virus titers were lower in calves with mixed infection. An increase in serum antibodies to both BHV-1 and PIV-3 was detected by serum neutralization and enzyme-linked immunosorbent assay. Antibody responses were delayed and significantly lower in calves given mixed infection than in calves infected with a single virus. Interleukin-2 activity in cultures of lymphocytes from BHV-1 and BHV-1 plus PIV-3 infected calves was higher compared to control calves.     

The use of immunomodulators in the control of infectious bovine rhinotracheitis

Three experiments have been carried out to verify the effectiveness of an immunomodulator, Baypamun (Bayer AG) in limiting the spread of Bovine herpesvirus-1 (BHV-1), the causal agent of infectious bovine rhinotracheitis (IBR). In the first experiment, four calves infected with BHV-1 developed severe disease whereas four calves given Baypamun simultaneously with the virus had less severe disease. Four other calves in contact with the infected calves became severely ill but another four given Baypamun were only mildly affected. In the second experiment three calves infected with BHV-1, which reacted with typical disease, were allowed to remain in contact with six calves. All six calves were given Baypamun at various times following the exposure to BHV-1 infection and all showed a much reduced reaction with two treated for 4 days developing no clinical disease. Finally, in the third experiment one calf vaccinated one month before the start of the experiment did not develop any signs of disease when housed together with a calf experimentally infected with BHV-1. Of four other calves, vaccinated when the infected calf showed the first signs of disease, only the two given Baypamun in addition to the vaccine, were protected from clinical disease whereas the two given vaccine only developed classical signs of IBR. In the three experiments the virus shedding by the Baypamun-treated calves resulted to be significantly reduced.     

Influence of isoprinosine on bovine herpesvirus type-1 infection in cattle

A study was conducted to determine the in vivo efficacy of isoprinosine (ISO) in calves infected with bovine herpesvirus type-1 (BHV-1). Calves were infected with BHV-1 on day 0 and received ISO daily for 14 days. Clinical signs of disease, shedding of BHV-1, lymphocyte proliferative responses to mitogens, interleukin-2 production, and alveolar macrophage bactericidal activity were monitored during the study. Rectal temperatures were increased (P less than 0.05) in BHV-1 and ISO-BHV-1 calves at days 3 to 7 postinfection (PI). Isoprinosine did not influence BHV-1 shedding in calves. Lymphocyte proliferative responses to phytohemagglutinin (PHA) were lower (P less than 0.01) in BHV-1 calves when compared to control or ISO calves at day 4 PI, but ISO did not ameliorate this effect. Interleukin-2 activity was greater (P less than 0.05) in ISO-BHV-1 calves on days 4 and 8 PI in PHA-stimulated lymphocytes and on day 8 PI in concanavalin A-stimulated lymphocytes when compared to control, ISO or BHV-1 calves. Isoprinosine treatment of BHV-1-infected calves tended to decrease alveolar macrophage bactericidal activity. These data suggest that ISO does not reverse BHV-1 suppression of lymphocyte proliferation, but may enhance IL-2 production in BHV-1 infected calves.     

Attempts to reactivate bovid herpesvirus-2 in experimentally infected calves

A study was carried out to determine whether bovid herpesvirus-2 (BHV-2) is able to induce a recurrent infection in experimentally infected calves. Twelve calves infected with the virus were treated with dexamethasone (DMS) beginning 69 days after the infection, ie, several weeks after the animals had recovered from the disease and were negative for BHV-2. The stress induced by DMS treatment failed to reactivate the clinical condition or to induce shedding of BHV-2. However, treatment with DMS reactivated a latent infectious bovine rhinotracheitis (IBR) virus infection in all calves previously inoculated with BHV-2, and also in 2 noninoculated controls. The reactivation of IBR virus occurred without any clinical evidence of the disease, but the virus was isolated from nasal and pharyngeal swabbings and from the organs. A proliferative ganglionitis of the trigeminal ganglion was also observed. Because of the interference by IBR virus, this study did not resolve the question as to whether BHV-2 can induce a recurrent infection.     

DNA of bovine herpesvirus type 1 in the trigeminal ganglia of latently infected calves

Twelve calves infected with bovine herpesvirus type 1 (BHV-1) were killed when in a latent state of infection. Latency was verified 30 days after virus inoculation of the calves by seroconversion, absence of virus shedding, and in 2 calves, by recrudescence of the infection after they were treated with dexamethasone. By in situ hybridization techniques and autoradiography, DNA of BHV-1 was detected in 13 of 23 trigeminal ganglia of latently infected calves. Viral DNA was restricted to the nucleus of nerve cells. Single neurons harboring BHV-1 DNA were observed in 4.9% of the sections (n = 325) of the trigeminal ganglia. The results obtained correspond to those known from herpes simplex virus infections in mice. The implications for the virus-host relationship are discussed.     

Periodic reappearance of bovine herpesvirus type 4 DNA in the sera of naturally and experimentally infected rabbits and calves.

A BHV-4 specific nested PCR was used for the detection of viral DNA in serum samples of rabbits and calves. All animals were followed up for 62 days, blood samples were taken for PCR studies every second day. Maternal infection of calves resulted in the repeated regular reappearance (10-14 days) of the virus (DNA) in serum samples. When PCR positive five-day-old calves were infected with tissue culture adapted virus, the reappearance of the DNA in the serum was shown to be irregular, nevertheless, DNA peaks reappeared during the whole observation period. A PCR negative calf infected at the age of 60 days was found to possess viraemia until p.i.d. 32. In rabbits treated intravenously with BHV-4 the inoculum or a primary viraemia was detected at p.i.d. 2-3 and p.i.d. 14-16. Published data on human herpesviruses suggest, that the target cells might be a pluripotent stem cell population of the bone marrow and differentiated virus-infected cells destroyed by the immune system might be the source of viral DNA detected in the serum. Frequency of DNA reappearance was depended on the age of the infected animals but not on the inoculated amount of BHV-4. The described phenomenon might be part of BHV-4 infection of very young animals.     

Immunoglobulin M (IgM) indirect enzyme-linked immunosorbent assay and the involvement of IgM-rheumatoid factor in the serodiagnosis of BHV-1 infection.

An IgM enzyme-linked immunosorbent assay (IgM-ELISA) for the detection of antibodies to bovine herpesvirus-1 (BHV-1) was developed. Its applicability was examined by serological studies in two calves experimentally infected with virulent BHV-1 over a period of 60 days. IgM antibodies were detected by ELISA on day 6 after infection, and there was an increase in IgG antibodies on day 9. Serum neutralizing (SN) antibodies were detected only on day 13, confirming the higher sensitivity of the ELISAs. A similar study of four calves treated with a commercial inactivated virus vaccine indicated no detectable IgM-ELISA response, and late SN and IgG-ELISA reactivity. Thus IgM-ELISA appears to be of value in assessing recent infection, whereas IgG-ELISA and SN cannot distinguish between infection and vaccination. The possible limitations imposed on the specific IgM-ELISA by the presence of IgM rheumatoid factor (IgM-RF) in bovine serum were examined. IgM-RF levels were determined in bovines of various ages. Elevated values of IgM-RF were found in the sera of older animals; their occurrence may lead to false IgM-positive diagnosis (16%) of BHV-1 infection. This was examined in 113 serum specimens from suspected cases of BHV-1 infection and in 32 bulls at an insemination center. Pretreatment of serum samples with an antibovine IgG serum eliminated false positivity of the IgM-ELISA. It is concluded that IgM-ELISA should be of particular value in the diagnosis of recent infection with BHV-1, mainly in calves.     

Response of proinflammatory and anti-inflammatory cytokines in calves with subclinical bovine viral diarrhea challenged with bovine herpesvirus-1

The aim of this work was to investigate the susceptibility of calves infected with bovine viral diarrhea virus (BVDV) against secondary infections. For this purpose, the profile of cytokines implicated in the immune response of calves experimentally infected with a non-cytopathic strain of BVDV type-1 and challenged with bovine herpesvirus 1.1 (BHV-1.1) was evaluated in comparison with healthy animals challenged only with BHV-1.1. The immune response was measured by serum concentrations of cytokines (IL-1β, TNFα, IFNγ, IL-12, IL-4 and IL-10), acute phase proteins (haptoglobin, serum amyloid A and fibrinogen) and BVDV and BHV-1.1 specific antibodies. BVDV-infected calves displayed a great secretion of TNFα and reduced production of IL-10 following BHV-1 infection, leading to an exacerbation of the inflammatory response and to the development of more intense clinical symptoms and lesions than those observed in healthy animals BHV-1-inoculated. A Th1 immune response, based on IFNγ production and on the absence of significant changes in IL-4 production, was observed in both groups of BHV-1-infected calves. However, whereas the animals inoculated only with BHV-1 presented an IFNγ response from the start of the study and high expression of IL-12, the BVDV-infected calves showed a delay in the IFNγ production and low levels of IL-12. This alteration in the kinetic and magnitude of these cytokines, involved in cytotoxic mechanisms responsible for limiting the spread of secondary pathogens, facilitated the dissemination of BHV-1.1 in BVDV-infected calves.     

Recrudescence of bovine herpesvirus-5 in experimentally infected calves

A latent infection of bovine herpesvirus-5 (BHV-5) was established in 4 calves. These calves, plus 2 controls, were given dexamethasone (DM) to reactivate the latent virus. The 4 principal calves developed antibodies to BHV-5 by postinoculation day (PID) 21. Antibody titers increased until PID 42 before decreasing to low levels of PID 75. After the first DM treatment (started on PID 76), an anamnestic antibody response was demonstrated in the 4 principal calves. Calves, 2, 3, and 4 were euthanatized and necropsied at PID 121, and their antibody titers were again decreasing. The virus BHV-5 was not isolated from the tissues by conventional techniques of viral isolation but was isolated from the trigeminal ganglion and spinal cord of calf 3 by explantation techniques. The BHV-5 was isolated, using conventional viral isolation techniques, from a nasal swab sample of calf 1 on PID 91 (15 days after the first DM treatment) and from the thoracic lymph node 6 days after the start of a 2nd DM treatment. Seemingly, BHV-5 may be latently harbored in the nerve tissues or calves and this virus may be reactivated from the upper respiratory tract following subsequent DM treatment.     

Fatal, generalized bovine herpesvirus type-1 infection associated with a modified-live infectious bovine rhinotracheitis parainfluenza-3 vaccine administered to neonatal calves.

Generalized bovine herpesvirus 1 (BHV-1) infection was diagnosed in six Salers calves from the same herd. The calves had received an intramuscular injection of modified-live infectious bovine rhinotracheitis parainfluenza-3 vaccine between birth and three days of age. The purpose of this study was to determine if the outbreak was associated with the vaccine strain of BHV-1. Analysis of epidemiological data and BHV-1 DNA for restriction fragment length polymorphism was undertaken. Multifocal necrosis in multiple organs was observed on pathological examination, and the presence of BHV-1 in tissues was confirmed by immunohistochemistry. Forty-three calves (aged birth to thirty days) were vaccinated over an 11-day interval. The 10 deaths recorded for vaccinated calves were clustered over a subsequent 14-day interval. Mortality in calves vaccinated between birth and three days of age was significantly higher than in nonvaccinated calves (chi-square test; p < or = 0.025), and this mortality was characterized by a greater age at death and duration of illness for vaccinated calves (t test; p < or = 0.001). The patterns of the restriction fragments, generated by six restriction endonucleases, of BHV-1 isolated from a necropsied calf and from the vaccine were identical, and different from that of a laboratory strain of BHV-1 (P8-2). These findings support the conclusion that newborn calves were susceptible to an intramuscularly injected vaccine strain of BHV-1, and that administration of an intramuscular modified-live infectious bovine rhinotracheitis parainfluenza-3 vaccine to neonatal calves may not be an innocuous procedure.     

Antibody response to glycoprotein E after bovine herpesvirus type 1 infection in passively immunised, glycoprotein E-negative calves

This study was conducted to determine whether young calves with maternal antibodies against bovine herpesvirus type 1 (BHV-1) but without antibodies against glycoprotein E (gE) can produce an active antibody response to gE after a BHV-1 infection. Five calves received at birth colostrum from gE-seronegative cows which had been vaccinated two or three times with an inactivated BHV-1, gE-deleted marker vaccine. After inoculation with a wild-type virulent strain of BHV-1, all the passively immunised gE-negative calves shed virus in large amounts in their nasal secretions. All the calves seroconverted to gE within two to four weeks after inoculation and then had high levels of gE antibodies for at least four months. The development of an active cell-mediated immune response was also detected by in vitro BHV-1-specific interferon-gamma assays. All the calves were latently infected, because one of them re-excreted the virus spontaneously and the other four did so after being treated with dexamethasone. The results showed that under the conditions of this work the gE-negative marker could also distinguish between passively immunised and latently infected calves.     

Effects in calves of mixed infections with bovine viral diarrhea virus and several other bovine viruses

The objective of this study was to verify whether a mixed infection in calves with bovine viral diarrhea virus (BVDV) and other bovine viruses, such as bovid herpesvirus-4 (BHV-4), parainfluenza-3 (PI-3) and infectious bovine rhinotracheitis (IBR) virus, would influence the pathogenesis of the BVDV infection sufficiently to result in the typical form of mucosal disease being produced.

Accordingly, two experiments were undertaken. In one experiment calves were first infected with BVDV and subsequently with BHV-4 and IBR virus, respectively. The second experiment consisted in a simultaneous infection of calves with BVDV and PI-3 virus or BVDV and IBR virus.

From the first experiment it seems that BVDV infection can be reactivated in calves by BHV-4 and IBR virus. Evidence of this is that BVDV, at least the cytopathic (CP) strain, was recovered from calves following superinfection. Moreover, following such superinfection the calves showed signs which could most likely be ascribed to the pathogenetic activity of BVDV. Superinfection, especially by IBR virus, created a more severe clinical response in calves that were initially infected with CP BVDV, than in those previously given the non-cytopathic (NCP) biotype of the virus. Simultaneous infection with PI-3 virus did not seem to modify to any significant extent the pathogenesis of the experimentally induced BVDV infection whereas a severe clinical response was observed in calves when simultaneous infection was made with BVDV and IBR virus.     

Longevity of protective immunity to experimental bovine herpesvirus-1 infection following inoculation with a combination modified-live virus vaccine in beef calves

OBJECTIVE: To determine whether a combination viral vaccine containing modified-live bovine herpesvirus-1 (BHV-1) would protect calves from infection with a recent field isolate of BHV-1. DESIGN: Randomized controlled trial. ANIMALS: Sixty 4- to 6-month-old beef calves. PROCEDURE: Calves were inoculated with a placebo 42 and 20 days prior to challenge (group 1; n = 10) or with the combination vaccine 42 and 20 days prior to challenge (group 2; 10), 146 and 126 days prior to challenge (group 3; 10), 117 and 96 days prior to challenge (group 4; 10), 86 and 65 days prior to challenge (group 5; 10), or 126 days prior to challenge (group 6; 10). All calves were challenged with BHV-1 via aerosol. Clinical signs, immune responses, and nasal shedding of virus were monitored for 14 days after challenge. RESULTS: Vaccination elicited increases in BHV-1-specific IgG antibody titers. Challenge with BHV-1 resulted in mild respiratory tract disease in all groups, but vaccinated calves had less severe signs of clinical disease. Extent and duration of nasal BHV-1 shedding following challenge was significantly lower in vaccinated calves than in control calves. In calves that received 2 doses of the vaccine, the degree of protection varied with the interval between the last vaccination and challenge, as evidenced by increases in risk of clinical signs and extent and duration of viral shedding. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that this combination vaccine provided protection from infection with virulent BHV-1 and significantly reduced nasal shedding of the virus for at least 126 days after vaccination.     

Bovine herpesvirus type 1 (BHV-1) mutant lacking U(L)49.5 luminal domain residues 30-32 and cytoplasmic tail residues 80-96 induces more rapid onset of virus neutralizing antibody and cellular immune responses in calves than the wild-type strain Cooper

Bovine herpesvirus type 1 (BHV-1) envelope protein U(L)49.5 inhibits transporter associated with antigen processing (TAP) and down-regulates cell-surface expression of major histocompatibility complex (MHC) class I molecules to promote immune evasion. Earlier, we have constructed a BHV-1U(L)49.5Δ30-32 CT-null virus and determined that in the infected cells, TAP inhibition and MHC-I down regulation properties of the virus are abolished. In this study, we compared the pathogenicity and immune responses in calves infected with BHV-1U(L)49.5Δ30-32 CT-null and BHV-1 wt viruses. Following primary infection, both BHV-1 wt and BHV-1U(L)49.5Δ30-32 CT-null virus replicated in the nasal epithelium with very similar yields. BHV-1 antigen-specific CD8+ T cell proliferation as well as CD8+ T cell cytotoxicity in calves infected with the BHV-1U(L)49.5Δ30-32 CT-null virus peaked by 7 dpi (P<0.05) which is 7 days earlier than that of BHV-1 wt-infected calves. Further, virus neutralizing antibody (VN Ab) titers and IFN-γ producing peripheral blood mononuclear cells (PBMCs) in the U(L)49.5 mutant virus-infected calves, also peaked 7 days (IFN-γ; P<0.05) and 14 days (VN Ab; P<0.05) earlier, respectively. Therefore, relative to wt in the BHV-1U(L)49.5 mutant virus-infected calves, primary neutralizing antibody and cellular immune responses were induced significantly more rapidly.     

Experimental infection of calves with strains of Bovid herpesvirus-4

Fourteen calves were inoculated intranasally (i.n.) with the viral isolates as follows: 5 with 85/BH 16TV, 1 with 85/BH 17TV, 1 with 85/BH 18TV, 2 with 85/BH 231TN and 5 with 85/BH 232TN. Strain 85/BH 16TV was the only one which caused overt respiratory-like disease in all inoculated calves. Onset of the disease was observed after 7-8 days of incubation and was characterized by fever, depression, nasal discharge and coughing. Virus was isolated from the nasal swabbings of calves obtained from post-infection day (PID) 2-10. The other viral strains did not cause any sign of disease although virus was isolated regularly from the nasal swabbings of the inoculated calves. Virus was recovered from central nervous system tissues of calves that were infected with 85/BH 16TV or 85/BH 232TN strains and were killed on PID 4 or 8. Virus was also isolated from other tissues, such as lymph node, nasal mucosa (PID 8), or lung (PID 4). It was speculated that the nervous system could be one of the target areas of the virus of the naturally occurring infection by BHV-4. This might indicate a possible role of the nervous system (site of latency?) in the pathogenesis of BHV-4 as is the case in certain herpesviral infections of man and the lower animals.     

Experimental latent and recrudescent bovine herpesvirus-1 infections in calves

Latent bovine herpesvirus-1 (BHV-1) infection was established in 6 calves and was demonstrated by reinduction of virus shedding after administration of corticosteroids. Latently infected calves failed to transmit BHV-1 during 4 weeks' contact with sentinel calves. Infected calves were killed and necropsied during latency or induced recrudescence. The BHV-1 DNA was demonstrated intranuclearly in trigeminal ganglion neurons by in situ hybridization. The BHV-1 antigen was demonstrated by immunofluorescence in trigeminal ganglion neurons during recrudescence. By electron microscopy, changes in the appearance of the Nissl bodies and a high frequency of nuclear bodies were observed in trigeminal ganglion neurons.     

Detection of BHV-1 in a Naturally Infected Bovine Fetus by a Nested PCR assay

Bovine herpesvirus 1 (BHV-1) is frequently associated with abortion in naturally and experimentally infected cattle. Most of the virus isolation and immunofluorescent antibody protocols described in the literature for detecting BHV-1 in bovine foetuses are rather laborious, costly and time-consuming. The detection is described of BHV-1 in the tissues of a naturally aborted bovine foetus by a nested PCR assay with no further hybridization procedures.Optimal results were achieved by filtering the foetal tissues on a chromatography column before DNA extraction, by using two pairs of primers in a nested PCR and by evaluating the amplification products on silver-stained polyacrylamide gels.This nested PCR was faster and easier to perform than the virus isolation test. To our knowledge, this is the first time that BHV-1 has been detected in the tissues of a naturally infected bovine foetus by means of a nested PCR. The test seems to be a practical alternative for rapid detection of BHV-1 in bovine foetus.     

Effects of a bovine herpesvirus-1 isolate on reproductive function in heifers: classification as a type-2 (infectious pustular vulvovaginitis) virus by restriction endonuclease analysis of viral DNA

A bovine herpesvirus-1 (BHV-1) isolate (FI) from an aborted fetus was used to infect 9 heifers at various stages of gestation. Two heifers were inoculated IV on postbreeding day (PBD) 1, 7, or 14, and 3 heifers were inoculated in the sixth month of pregnancy. Plasma progesterone assays were used to monitor corpus luteum function in heifers inoculated during early pregnancy. Low progesterone values and infertility were seen in the 2 heifers inoculated on PBD 1. Luteal function remained normal in heifers inoculated on PBD 7 or 14. These 4 heifers inoculated on PBD 7 or 14 carried their fetuses to term, and their calves were free of BHV-1 infection at birth. Three heifers inoculated during the sixth month of pregnancy also carried their fetuses to term. Two calves were born alive, and BHV-1 was not isolated from nasal swab samples of either calf; the third calf was stillborn. Virus was not isolated from the stillborn calf's tissues, but BHV-1 was isolated from the placenta. Lesions were not detected in several tissues examined by light microscopy, and BHV-1 antigen was not detected by immunohistochemical examination of paraffin sections. Restriction endonuclease analysis of viral DNA was used to compare the FI virus to other BHV-1 isolates (Colorado-1, Iowa, and K22). On the basis of restriction endonuclease analysis, the FI isolate should be classified as a type-2 (infectious pustular vulvovaginitis) virus, specifically subtype a.     

Analysis of bovine trigeminal ganglia following infection with bovine herpesvirus 1

Following primary infection of the eye, oral cavity, and/or nasal cavity, bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic (TG) neurons. Virus reactivation and spread to other susceptible animals occur after natural or corticosteroid-induced stress. Infection of calves with BHV-1 leads to infiltration of lymphocytes in TG and expression of IFN-gamma (interferon-gamma), even in latently infected calves. During latency, virus antigen and nucleic acid positive non-neural cells were occasionally detected in TG suggesting there is a low level of spontaneous reactivation. Since we could not detect virus in ocular or nasal swabs, these rare cells do not support high levels of productive infection and virus release or they do not support virus production at all. Dexamethasone (DEX) was used to initiate reactivation in latently infected calves. Foci of mononuclear or satellite cells undergoing apoptosis were detected 6h after DEX treatment, as judged by the appearance of TUNEL+ cells (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling). BHV-1 antigen expression was initially detected in lymphocytes and other non-neural cells in latently infected calves following DEX treatment. At 24h after DEX treatment, viral antigen expression and nucleic acid were readily detected in neurons. Our data suggest that persistent lymphocyte infiltration and cytokine expression occur during latency because a low number of cells in TG express BHV-1 proteins. Induction of apoptosis and changes in cytokine expression following DEX treatment correlates with reactivation from latency. We hypothesize that inflammatory infiltration of lymphoid cells in TG plays a role in regulating latency.     

Dynamics of Moraxella bovis infection and humoral immune response to bovine herpes virus type 1 during a natural outbreak of infectious bovine keratoconjunctivitis in beef calves

Infectious bovine keratoconjunctivitis (IBK) is an acute disease caused by Moraxella bovis (Mb). Several factors may predispose animals to an IBK outbreak; one commonly observed is infection with bovine herpes virus type 1 (BHV-1). The aim of this study was to investigate the dynamics of BHV-1 virus infection and its relation with clinical cases of IBK in weaned calves from a beef herd with a high prevalence of lesions caused by Mb. Sampling was carried out in six stages and included conjunctival swabs for isolating Mb as well as blood samples for identifying antibodies specific for BHV-1. A score for IBK lesions after observing each eye was determined. The findings of this study showed a high prevalence of BHV-1 virus infection (100% of animals were infected at the end of the trial); 67% of animals were culture-positive for Mb, but low rates of clinical IBK (19% of calves affected) were detected at the end of the trial. These results suggest that infection with BHV-1 did not predispose these animals to IBK, and that Mb infection produced clinical and subclinical disease in the absence of BHV-1 co-infection.