Efficacy of a modified live intranasal bovine respiratory syncytial virus vaccine in 3-week-old calves experimentally challenged with BRSV

Two experimental bovine respiratory syncytial virus (BRSV) challenge studies were undertaken to evaluate the efficacy of a single intranasal dose of a bivalent modified live vaccine containing BRSV in 3-week-old calves. In the first study, vaccine efficacy was evaluated in colostrum deprived (maternal antibody negative) calves 5, 10 and 21 days after vaccination. Nasal shedding of BRSV was significantly reduced in vaccinated calves challenged 10 or 21 days after vaccination. Virus excretion titres were also reduced in vaccinates challenged 5 days after vaccination but reduction in duration of shedding and total amount of virus shed were not statistically significant. Clinical disease after challenge in this study was mild. In the second study, vaccine efficacy was assessed in calves with maternal antibodies against BRSV by challenge 66 days post-vaccination. Vaccination significantly reduced nasal shedding after challenge and the severity of clinical disease was also reduced.     

Efficacy of a saponin-adjuvanted inactivated respiratory syncytial virus vaccine in calves

The objective of this study was to determine whether a commercially available, saponin-adjuvanted, inactivated bovine respiratory syncytial virus (BRSV) vaccine would protect calves from experimental infection with virulent BRSV. This was a randomized controlled trial comprising 14, 8- to 9-week-old calves seronegative for BRSV Group 1 calves (n = 8) were not vaccinated and group 2 calves (n = 6) were vaccinated on days 0 and 19 with an inactivated BRSV vaccine. All calves were challenged with virulent BRSV on day 46. Clinical signs, arterial PO2, and immune responses were monitored after challenge. Calves were euthanatized on day 54 (8 d after challenge) and lungs were examined for lesions. Vaccination elicited increases in BRSV-specific immunoglobulin (Ig) G and virus neutralizing antibody titers. Challenge with BRSV resulted in severe respiratory tract disease and extensive pulmonary lesions in control calves, but no signs of clinical disease and minimal or no pulmonary lesions in vaccinated calves. Arterial blood oxygen values on day 53 (7 d after challenge) in control calves were significantly lower than those in vaccinated calves, which remained within normal limits. Control calves shed BRSV for several days after challenge, whereas BRSV was not detected on deep nasal swabs from vaccinated calves. In summary, the results indicated that this inactivated BRSV vaccine provided clinical protection from experimental infection with virulent virus 27 d after vaccination and significantly decreased the prevalence and severity of pulmonary lesions. Efficacy was similar to that reported for other commercial inactivated and modified-live BRSV vaccines.     

Efficacy of an intranasal modified live bovine respiratory syncytial virus and temperature-sensitive parainfluenza type 3 virus vaccine in 3-week-old calves experimentally challenged with PI3V

Two experimental parainfluenza type 3 virus (PI3V) challenge studies were undertaken to evaluate the efficacy of a single intranasal dose of an attenuated live vaccine containing modified live bovine respiratory syncytial virus (BRSV) and temperature-sensitive PI3V in 3-week-old calves. In the first study, vaccine efficacy was evaluated in colostrum deprived calves. Nasal shedding of PI3V was highly significantly reduced in vaccinated calves challenged 10 days or 21 days after vaccination. In the second study, vaccine efficacy was assessed in calves with maternal antibodies against PI3V by challenge 66 days post-vaccination. Vaccination also significantly reduced PI3V excretion after challenge in this study. In both studies, clinical signs after challenge were very mild and were not different between vaccinated and control calves.     

Efficacy of an inactivated respiratory syncytial virus vaccine in calves.

OBJECTIVE: To determine whether an inactivated bovine respiratory syncytial virus (BRSV) vaccine would protect calves from infection with virulent BRSV. DESIGN: Randomized controlled trial. ANIMALS: 27 nine-week-old calves seronegative for BRSV exposure. PROCEDURE: Group-1 calves (n = 9) were not vaccinated. Group-2 calves (n = 9) were vaccinated on days 0 and 21 with an inactivated BRSV vaccine containing a minimum immunizing dose of antigen. Group-3 calves (n = 9) were vaccinated on days 0 and 21 with an inactivated BRSV vaccine containing an amount of antigen similar to that in a commercial vaccine. All calves were challenged with virulent BRSV on day 42. Clinical signs and immune responses were monitored for 8 days after challenge. Calves were euthanatized on day 50, and lungs were examined for lesions. RESULTS: Vaccination elicited increases in BRSV-specific IgG and virus neutralizing antibody titers and in production of interferon-gamma. Virus neutralizing antibody titers were consistently less than IgG titers. Challenge with BRSV resulted in severe respiratory tract disease and extensive pulmonary lesions in control calves, whereas vaccinated calves had less severe signs of clinical disease and less extensive pulmonary lesions. The percentage of vaccinated calves that shed virus in nasal secretions was significantly lower than the percentage of control calves that did, and peak viral titer was lower for vaccinated than for control calves. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the inactivated BRSV vaccine provided clinical protection from experimental infection with virulent virus and decreased the severity of pulmonary lesions. Efficacy was similar to that reported for modified-live BRSV vaccines.     

T-cell populations responsive to bovine respiratory syncytial virus in seronegative calves

Calves lacking detectable serum antibodies against bovine respiratory syncytial virus (BRSV) were screened for virus-specific T-cell memory. Peripheral blood mononuclear cells were cultured in vitro with live BRSV and analyzed by dual-color flow cytometry for surface expression of CD25 on CD4(+), CD8(+), and gammadeltaT-cells. Significant recall responses were detected in some of the seronegative calves. Modified live BRSV vaccine was administered to these and to a group of non-responding calves. Following vaccination, virus-specific IgG, virus neutralizing antibody, and T-cell recall responses were all elevated more rapidly in the group with BRSV-sensitive T-cells than in the T-cell-negative group, which suggested that calves in the first group were previously exposed to BRSV. This demonstrates that exposure to BRSV can induce T and B cell memory in young calves without causing seroconversion. The calves were presumably exposed to BRSV while they had maternal antibody, which inhibited the calves from developing an antibody response.     

Bovine Para-Influenza 3 Vaccine Studies

A bovine para-influenza 3 modified live virus vaccine was studied in colostrum-deprived calves. A single dose of vaccine induced antibody responses and afforded protection against experimental challenge. This was evidenced by reduced clinical disease and virus excretion as compared to unvaccinated control calves. The vaccine virus did not spread from vaccinated calves to contact controls.     

Response of calves to challenge exposure with virulent bovine respiratory syncytial virus following intranasal administration of vaccines formulated for parenteral administration

OBJECTIVE: To determine whether single-fraction and combination modified-live bovine respiratory syncytial virus (BRSV) vaccines commercially licensed for parenteral administration could stimulate protective immunity in calves after intranasal administration. DESIGN: Randomized controlled trial. ANIMALS: 39 calves. PROCEDURES: Calves were separated from dams at birth, fed colostrum with a minimal concentration of antibodies against BRSV, and maintained in isolation. In 2 preliminary experiments, 9-week-old calves received 1 (n = 3) or 2 (3) doses of a single-component, modified-live BRSV vaccine or no vaccine (8 control calves in each experiment), and were challenged with BRSV 21 days after vaccination. In a third experiment, 2-week-old calves received combination modified-live virus (MLV) vaccines with or without BRSV and calves were challenged with BRSV 8 days later. Calves were euthanized, and lung lesions were measured. Immune responses, including serum and nasal antibody and nasal interferon-alpha concentrations, were assessed. RESULTS: BRSV challenge induced signs of severe clinical respiratory tract disease, including death and pulmonary lesions in unvaccinated calves and in calves that received a combination viral vaccine without BRSV. Pulmonary lesions were significantly less severe in BRSV-challenged calves that received single or combination BRSV vaccines. The proportion of calves that shed virus and the peak virus titer was decreased, compared with control calves. Protection was associated with mucosal IgA antibody responses after challenge. CONCLUSIONS AND CLINICAL RELEVANCE: Single and combination BRSV vaccines administered intranasally provided clinical protection and sparing of pulmonary tissue similar to that detected in response to parenteral delivery of combination MLV and inactivated BRSV vaccines previously assessed in the same challenge model.     

Effects of a single intranasal dose of modified-live bovine respiratory syncytial virus vaccine on resistance to subsequent viral challenge in calves

OBJECTIVE: To determine whether a single intranasal dose of modified-live bovine respiratory syncytial virus (BRSV) vaccine protects calves from BRSV challenge and characterize cell-mediated immune response in calves following BRSV challenge. ANIMALS: 13 conventionally reared 4- to 6-week-old Holstein calves. PROCEDURES: Calves received intranasal vaccination with modified live BRSV vaccine (VC-group calves; n = 4) or mock vaccine (MC-group calves; 6) 1 month before BRSV challenge; unvaccinated control-group calves (n = 3) underwent mock challenge. Serum virus neutralizing (VN) antibodies were measured on days -30, -14, 0, and 7 relative to BRSV challenge nasal swab specimens were collected for virus isolation on days 0 to 7. At necropsy examination on day 7, tissue specimens were collected for measurement of BRSV-specific interferon gamma (IFN-gamma) production. Tissue distribution of CD3+ T and BLA.36+ B cells was evaluated by use of immunohistochemistry. RESULTS: The MC-group calves had significantly higher rectal temperatures, respiratory rates, and clinical scores on days 5 to 7 after BRSV challenge than VC-group calves. No difference was seen between distributions of BRSV in lung tissue of VC- and MC-group calves. Production of BRSV-specific IFN-gamma was increased in tissue specimens from VC-group calves, compared with MC- and control-group calves. Virus-specific IFN-gamma production was highest in the mediastinal lymph node of VC-group calves. Increased numbers of T cells were found in expanded bronchial-associated lymphoid tissue and airway epithelium of VC-group calves. CONCLUSIONS AND CLINICAL RELEVANCE: An intranasal dose of modified-live BRSV vaccine can protect calves against virulent BRSV challenge 1 month later.     

Antibody response of calves to immunoaffinity-purified bovine respiratory syncytial virus VP70 after vaccination and challenge exposure.

Immunoaffinity-purified bovine respiratory syncytial virus (BRSV) fusion (F) protein elicited anti-BRSV-specific antibody responses in BRSV-seronegative calves. After primary vaccination, all calves seroconverted to BRSV as determined by the virus neutralization (VN) test and developed anti-F protein antibodies detectable by protein immunoblot analyses. Subsequent vaccinations induced greater than twofold increase in VN titer in 3 of 9 (33%) calves, and 1 calf became VN-negative, but still had nonneutralizing antibody detectable by protein immunoblot analysis. This calf remained seronegative after challenge exposure. Two groups of calves were vaccinated IM with immunoaffinity-purified BRSV F protein. Each dose was 2 ml containing 20 micrograms of purified F protein. Freund's adjuvants were used for all vaccinations, with Freund's complete adjuvant used for the primary vaccination and Freund's incomplete adjuvant for subsequent vaccinations. The vaccine was administered to both groups at weeks 0 and 3; the first group received a third vaccination at weeks 21. Group-1 and -2 vaccinated calves and non-vaccinated contact controls were intranasally aerosol challenge-exposed with low cell culture-passage BRSV on weeks 22 and 9, respectively. Eight of 9 vaccinated calves did not develop a humoral anamnestic response following challenge exposure, as demonstrated by VN test and protein immunoblot analyses. Calf 14 from group 1 which had a 1:2 VN antibody titer prior to vaccination, was the only calf that developed an anamnestic response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis of antibody responses of feedlot cattle to bovine respiratory syncytial virus following vaccination with mixed vaccines.

The antibody response of cattle to bovine respiratory syncytial virus (BRSV) immunization was investigated using 4 different commercially available mixed vaccines. Forty, 5-6 month old, beef calves, randomly assigned to groups of 10, were vaccinated on day 0 and 21 with 1 of 3 inactivated vaccines, (3 groups), or a modified live virus (MLV) vaccine. BRSV-specific antibody responses were measured prior to vaccination and on day 35 by using an enzyme linked immunosorbent assay (ELISA), virus neutralization assay (VN), a fusion inhibition assay (FI); and responses were also measured for their ability to facilitate antibody dependent, complement mediated cytotoxicity (ADCMC) of BRSV infected cells. Sera from day 35 were, in addition, analyzed by use of an IgG1, IgG2 isotype specific ELISA. All vaccines induced significant increases in BRSV specific IgG antibody as measured by ELISA, but only one inactivated and the MLV vaccine induced significant increases in VN titers. Fusion inhibiting antibody titers were low or undetected in calves vaccinated with the inactivated vaccines. Vaccination with modified live virus induced significantly higher titers of fusion inhibiting antibodies, which are considered to be most highly correlated with protection. The VN to ELISA and FI to ELISA ratio of the calves that received MLV vaccine were significantly greater than the calves receiving the 3 inactivated vaccines. Vaccination with MLV induced the highest IgG2/IgG1 ratio. This difference was small, and only significant relative to 2 of the inactivated vaccine groups, which were not significantly different from each other. The higher proportion of IgG2 isotype in the MLV sera was not associated with lower ADCMC, a function not attributed to this isotype. The VN and FI titers, but not the ELISA value of the sera, were most predictive of ADCMC. The inactivation processes apparently alter epitopes and affect the induction of functional antibodies.     

Evaluation of efficacy of an inactivated vaccine against bovine respiratory syncytial virus in calves with maternal antibodies

OBJECTIVE: To assess short- and long-term efficacy of an inactivated bovine respiratory syncytial virus (BRSV) vaccine administered i.m. to calves with maternally derived antibodies. ANIMALS: 28 two-week-old calves with neutralizing, maternally derived antibodies against BRSV. PROCEDURE: For evaluation of short-term efficacy, 6 calves were vaccinated i.m. at 2 and 6 weeks of age and challenged intranasally and intratracheally along with a matched group of 4 unvaccinated control calves at 10 weeks of age. For evaluation of long-term efficacy, 2 groups of 6 calves each were vaccinated i.m. at 2, 6, and 18 weeks of age or 14 and 18 weeks of age; these calves were challenged intranasally and intratracheally along with 6 matched unvaccinated control calves at 43 weeks of age. Serum virus neutralizing antibody titer, clinical reactions, and virus shedding in nasal mucus and lung washings were assessed. RESULTS: None of the vaccination regimens resulted in a significant increase in serum virus neutralizing antibody titer. As judged by virus shedding in nasal mucus and lung washings, vaccinated calves were protected against challenge, compared with unvaccinated control groups. Clinical signs attributable to challenge were coughing (short-term efficacy study) and tachypnea and dyspnea (long-term efficacy study). The severity and incidence of disease were significantly lower in the vaccinated groups, compared with that in the unvaccinated groups. CONCLUSIONS AND CLINICAL RELEVANCE: Through vaccination, it is possible to protect vulnerable calves with maternal antibodies against BRSV infection and reduce respiratory tract disease.     

Role of IgE in the pathogenesis of bovine respiratory syncytial virus in sequential infections in vaccinated and nonvaccinated calves

A study was undertaken to evaluate the possible role of IgE in the pathogenesis of bovine respiratory syncytial virus (BRSV) infection. Fifteen calves were allotted at random to 3 treatment groups. One group of 6 calves was vaccinated with attenuated BRSV vaccine before live-virus challenge exposure, another group of 6 was not vaccinated before challenge exposure, and the remaining 3 calves served as controls (nonvaccinated, nonchallenge exposed). Calves of the 2 experimental groups were exposed to 2 live-virus aerosolizations (challenge exposure) 35 days apart. Histamine and BRSV-specific IgE (BRSV-IgE) concentrations in serum, lung lavage fluid, and nasopharyngeal exudate, as well as clinical signs of disease, were evaluated for 14 days after each challenge exposure. Vaccination before challenge exposure with live BRSV appeared to have little or no effect on the severity of the disease, but did appear to affect disease persistence. A correlation (P less than 0.02) existed between signs of disease and BRSV-IgE concentration measured in lung lavage fluid, but this was only true for vaccinated calves. Although no other correlations were found between clinical signs of disease and IgE concentration, analysis of the results additionally revealed a strong correlation (P less than 0.002) between disease signs and histamine concentration in nasopharyngeal exudate from calves of both experimental groups. Thus, indirect evidence implicated IgE in BRSV infection pathogenesis.     

The effect of dose, route and virulence of bovine herpesvirus 1 vaccine on experimental respiratory disease in cattle.

Three experiments were conducted with calves in which, following intramuscular or intranasal vaccination with virulent or attenuated bovine herpesvirus 1, calves were protected against bovine herpesvirus 1 -- Pasteurella haemolytica challenge. Calves receiving low doses of vaccine had lower levels of antibody and greater evidence of virus replication upon challenge than those receiving higher doses. In contrast 11/13 unvaccinated controls had fibrino-purulent pneumonia following challenge. The immune response developed later in younger calves and those given low doses of vaccine. Neutralizing antibodies to bovine herpes-virus 1 were not found in nasal secretions, but were present in serum seven days after vaccination. Bovine herpesvirus 1 was isolated before challenge from nasal secretions of calves vaccinated intranasally or intramuscularly with virulent virus but not those vaccinated intramuscularly with vaccine virus. It was concluded that both routes of vaccination with either virulent or attenuated bovine herpesvirus 1 provided protection from challenge with homologous or heterologous bovine herpesvirus 1 and that live vaccines should contain at least 10(3) plaque forming units/dose for effective immunization.     

Vaccination to protect calves against infectious bovine rhinotracheitis

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

An inactivated parainfluenza virus type 3 vaccine: the influence of vaccination regime on the response of calves and their subsequent resistance to challenge.

Calves maintained in insolated pens were vaccinated with an inactivated parainfluenza virus type (3) (pi3) vaccine usingparenteral and local route singly and in combination. The calves were subsequently monitored for serum antibody response and challenged intranasally with live virus to assess the protection derived from vaccination. Calves receiving one subcutaneous dose of vaccine in oil adjuvant produced a marked antibody response and were partially protected against challenge. Those receiving two successive subcutaneous doses produced a much greater antiboyd response and were completely protected against challenge. One intranasal dose of aqueous vaccine failed elicit a significant serum antibody response or protection against challenge. However, there was some evidence that intranasal vaccination following a single subcutaneous vaccination produced more effective immunity than one subcutaneous dose alone. Thus a vaccination regime was established which protected calves against experimental challenge and which could thefore be used in the field to assess the role of Pi3 virus in calf respiratory disease.     

Evaluation of a quadrivalent inactivated vaccine for the protection of cattle against diseases due to common viral infections

Efficacy of an inactivated quadrivalent vaccine containing infectious bovine rhinotracheitis (IBR) virus, parainfluenza type 3 (PI3) virus, bovine virus diarrhoea virus (BVDV) and bovine respiratory syncytial virus (BRSV) was assessed in naive bovine calves to evaluate short-term (4-18 weeks) and long-term (24-38 weeks) protection following the basic intramuscular vaccination regime of 2 inoculations a month apart. Vaccination was staggered between the long-term and the short-term groups by about 5 months so that both groups, along with a matched group of 6 unvaccinated (control) calves, could be challenged at the same time. Sequential challenges at intervals of 3-8 weeks were done in the order: IBR virus (intranasally, IN), PI3 virus (IN and intratracheally, IT), pestiviruses (IN) and BRSV (IN and IT). The IBR virus challenge produced febrile rhinotracheitis (FRT) in control calves but both the severity and the duration of FRT was significantly reduced in both vaccinated groups. The amount and the duration of IBR virus shed by the vaccinated groups was significantly reduced compared to the control group. Although PI3 virus, pooled pestivirus and BRSV challenges did not result in a noteworthy disease, challenge virus shedding (amount and duration) from the upper (all 3 viruses) and the lower (BRSV) respiratory tracts was significantly reduced in vaccinated groups. After pestivirus challenge, sera and leukocytes from all control calves were infectious for 6-9 days whereas virus was recovered only from leukocytes in vaccinated calves and only for 1.6-2.7 days. Thus a standard course of the quadrivalent vaccine afforded a significant protection against IBR virus, PI3 virus, BVDV and BRSV for at least 6 months.     

Inhibition of priming for bovine respiratory syncytial virus-specific protective immune responses following parenteral vaccination of passively immune calves

The effect of maternal antibodies (MatAb) on immunological priming by neonatal parenteral vaccination for bovine respiratory syncytial virus (BRSV) was addressed for the first time in experimental infection in 34 Holstein calves. Both vaccinated and control calves developed moderate to severe respiratory disease characteristic of acute BRSV infection. There were no differences in clinical signs, BRSV shed, arterial oxygen concentrations, or mortality between vaccinated and control calves after BRSV challenge approximately 11 wk after vaccination. There were no anamnestic antibody or cytokine responses in the vaccinates after challenge. Lung lesions were extensive in both groups, and although there was a statistically significant (P = 0.05) difference between groups, this difference was considered not biologically significant. These data indicate that stimulation of protective immune responses was inhibited by maternal antibodies when a combination modified-live BRSV vaccine was administered parenterally to young passively immune calves. Alternate routes of administration or different vaccine formulations should be used to successfully immunize young calves with good passive antibody transfer.     

Comparative serological response in calves to eight commercial vaccines against infectious bovine rhinotracheitis, parainfluenza-3, bovine respiratory syncytial, and bovine viral diarrhea viruses

A field trial was conducted to compare the serological responses in calves to eight commercial vaccines against infectious bovine rhinotracheitis virus (IBRV), parainfluenza-3 virus (PI3V), bovine respiratory syncytial virus (BRSV), and/or bovine viral diarrhea virus (BVDV). Calves given IBRV, P13V, BRSV, and BVDV vaccines had significantly higher antibodies to these viruses than unvaccinated controls; however, serological responses to killed BVDV vaccines were low. Calves with preexisting antibodies to IBRV, PI3V, BRSV, and the Singer strain of BVDV had lower seroconversion rates following vaccination than calves that were seronegative initially.

Serological responses in calves to IBRV, PI3V, BRSV, and BVDV differed among various commercial vaccines. Antibody titers to IBRV were higher in calves vaccinated with modified-live IBRV vaccines than in those vaccinated with killed IBRV vaccines. Following double vaccination with modified-live IBRV and PI3V vaccines, seroconversion rates and antibody titers to IBRV and PI3V were higher in calves vaccinated intramuscularly than in those vaccinated intranasally. Calves given Cattlemaster 4 had significantly higher titers to BRSV and PI3V, and lower titers to BVDV, than calves given Cattlemaster 3, suggesting that the addition of BRSV to Cattlemaster 4 caused some interaction among antigens.

     

Bovine respiratory syncytial virus-specific immune responses in cattle following immunization with modified-live and inactivated vaccines. Analysis of proliferation and secretion of lymphokines by leukocytes in vitro.

Cattle were immunized with vaccines containing modified-live or inactivated bovine respiratory syncytial virus (BRSV) and lymphocyte proliferative responses and cytokine secretion were monitored sequentially. Compared to pre-inoculated values, significant increases in proliferative responses to modified-live BRSV were detectable by Day 7 after the primary immunization with the vaccine containing inactivated BRSV, and by 7 days after the second immunization with modified-live virus. After a third immunization with the respective vaccines, proliferative responses to live BRSV were significantly higher in the group that received modified-live vaccine compared to the group that received inactivated vaccine. Proliferative responses to live BRSV corresponded with the presence of interleukin-2 (IL-2) in the supernatants from BRSV-stimulated leukocyte cultures and there were significantly higher levels of IL-2 in cultures from the group that received modified-live BRSV. An interferon species with the characteristics of interferon-alpha was also present in the supernatants from leukocyte cultures and there were no significant differences between the groups of vaccines. The predominant phenotype of proliferating cells in BRSV-stimulated leukocyte cultures derived from both groups of bovine vaccines was a BoCD4+ T-lymphocyte. These in vitro data suggest that both types of vaccines are capable of stimulating cell-mediated immune responses to BRSV in cattle.     

Enhancement of the immune response and virological protection of calves against bovine herpesvirus type 1 with an inactivated gE-deleted vaccine

Four immunisation protocols based on inactivated and attenuated commercially available marker vaccines for bovine herpesvirus type 1 (BHV-1) were compared. The first group of calves were vaccinated with an attenuated vaccine administered intranasally and an inactivated vaccine injected subcutaneously, four weeks apart; the second group were vaccinated twice with the attenuated vaccine, first intranasally and then intramuscularly; the third group were vaccinated twice subcutaneously with the inactivated vaccine; and the fourth group were vaccinated twice intramuscularly with the attenuated vaccine. A control group of calves were not vaccinated. The cellular and humoral immune responses were highest in the two groups which received at least one injection of the inactivated vaccine. Virological protection was observed in all the vaccinated groups after a challenge infection and reactivation by treatment with dexamethasone, but the calves which received one dose of the inactivated vaccine as a booster or two doses of the inactivated vaccine excreted significantly less of the challenge virus than the calves which were vaccinated only with the attenuated preparation.