Quantitation of hemorrhagic enteritis virus antigen and antibody using enzyme-linked immunosorbent assays.

Enzyme-linked immunosorbent assays (ELISAs) were developed to quantitate hemorrhagic enteritis virus (HEV) antibodies in turkey sera and HEV antigens in tissue extracts. These assays were more sensitive than the commonly used agar-gel precipitin tests in detecting antigen and antibody. The antibody-ELISA was used to monitor the presence and decline of passive antibodies in turkey poults and the seroconversion of turkeys infected with HEV. The antigen-ELISA was carried out using a monoclonal antibody; this test was used to quantitate HEV antigen in experimentally infected turkeys in a time-sequence experiment. Both ELISAs measured a strong antigenic relationship between an avirulent strain (HEV-A) and a virulent strain (HEV-V).

Propagation of group II avian adenoviruses in turkey and chicken leukocytes.

An avirulent hemorrhagic enteritis virus isolate (HEV-A) as well as a virulent one (HEV-V), both belonging to the group II avian adenoviruses, were successfully propagated in turkey leukocyte cell cultures. HEV antigens were detected as early as 12 hr after infection of the cells, using HEV-specific monoclonal antibodies in a fluorescent antibody test, and virus particles were observed by electron microscopy in the nuclei of infected cells at 18 to 24 hr after infection. Electron microscopy revealed the presence of HEV in the nuclei of nonadherent cells, as well as in adherent cells. The nonadherent infected cells had the characteristics of immature mononuclear leukocytes, whereas the adherent cells had monocyte-macrophage characteristics. HEV produced in turkey leukocytes was mostly cell-associated, particularly with the nonadherent cells. HEV-A could be serially passed in turkey blood leukocyte cultures at least seven times. Various methods employed to culture virus indicated that cells grown in spinner cultures were superior to cells grown in stationary cultures. In contrast to the successful infection of HEV in turkey leukocytes, the infection of chicken leukocytes with either HEV or splenomegaly virus of chickens, or turkey leukocytes with splenomegaly virus, was poor.

Efficacy of avirulent hemorrhagic enteritis virus propagated in turkey leukocyte cultures for vaccination against hemorrhagic enteritis in turkeys.

Avirulent hemorrhagic enteritis virus (HEV-A) propagated in turkey leukocyte cell culture was tested as a vaccine to prevent hemorrhagic enteritis (HE) in turkeys in experimental and field trials. Immunization of turkeys with live HEV-A resulted in protection against a challenge with virulent HEV (HEV-V) as measured by the serological response and the absence of clinical disease and HEV antigen in spleens. In field trials, 19 out of 20 flocks seroconverted within 21 days after vaccination with live HEV-A distributed in the drinking water. The average immune response of the turkeys in these flocks was 96%. Most importantly, neither clinical HE nor other adverse effects caused by HEV-A vaccination were observed in any of the vaccinated flocks. Because maternal antibodies can interfere with the immune response to the vaccine, the optimum time for vaccination was determined. Using an established half-life value of 4.25 days for turkey antibody, and knowing the enzyme-linked immunosorbent assay titer of the maternal antibodies and age of the turkey, the time of vaccination could be calculated taking into account that maternal antibody titers should be lower than 40 to vaccinate the turkeys successfully and induce protection. In vivo tests with HEV-A preparations confirmed the replication of the virus in turkey leukocyte cultures and the potential to pass it in culture. Furthermore, in vivo analysis showed that most infectious virus was associated with preparations of the nonadherent cell population, confirming the results obtained by in vitro analysis. The potency of HEV-A preparations was dependent on the production method and varied from an average of 570 to 8135 doses per ml.

Characterization of Escherichia coli isolated from cases of avian colibacillosis.

Forty-four western Canadian isolates of Escherichia coli associated with colibacillosis of turkeys and chickens were examined for serotype, antibiotic resistance, and production of aerobactin. The isolates belonged to fourteen O serogroups, with 39% of the strains being non-typeable. A high frequency of resistance to tetracycline, kanamycin, neomycin, cephalothin, streptomycin and erythromycin was observed. Most isolates produced aerobactin. Ten E. coli belonging to serogroups O1, O2 and O78 were also examined for pili production, hemagglutination, serum sensitivity, production of iron-regulated outer membrane proteins (IROMPS), and virulence. All isolates examined produced pili, exhibited mannose-sensitive hemagglutination of avian red blood cells and produced IROMPS under iron-restricted growth conditions. The five isolates of serogroup O1 and O2 were resistant to killing by turkey serum and were highly virulent. Only two of the five isolates of serogroup O78 were serum resistant. No correlation between serum resistance and virulence was observed in serogroup O78.

Characterization of group II avian adenoviruses with a panel of monoclonal antibodies.

The interaction between a panel of ten monoclonal antibodies and hemorrhagic enteritis virus, a group II avian adenovirus, was determined. The monoclonal antibodies reacted with all nine isolates of group II avian adenoviruses, but not with any of five types of group I avian adenoviruses. All ten monoclonal antibodies recognized antigenic determinants on the hexon protein of hemorrhagic enteritis virus when analyzed by immunoprecipitation and immunoblotting. They reacted only with the native hexon protein and not with protein denatured by sodium dodecyl sulfate or guanidine-HCl/urea treatment combined with reduction and carboxymethylation. Based on the results of competitive binding assays, the panel of monoclonal antibodies could be subdivided into two groups, which recognized different antigenic domains of the hemorrhagic enteritis virus hexon protein. The monoclonal antibodies in group 1 neutralized hemorrhagic enteritis virus infectivity while the monoclonal antibodies of group 2 did not. Group 1 consisted of eight monoclonal antibodies which could be further subdivided into subgroups 1A, 1B, 1C and 1D. The subdivision of the monoclonal antibodies was based on the degree of blocking in the competitive binding assays and differences in their ability to induce enhancement. In general, the monoclonal antibodies had a higher avidity for the virulent isolate of hemorrhagic enteritis virus than for the avirulent hemorrhagic enteritis virus isolate.

The prevalence of bovine viral diarrhea virus infection in a population of feedlot calves in western Canada.

The prevalence of bovine viral diarrhea virus (BVDV) infection was examined in a population of 5129 recently weaned steer calves entering a large feedlot in central Saskatchewan from September to December 1991. Serum samples were collected within 24 h of arrival at the feedlot from every fifth calf processed and again 96 d postarrival. A microtiter virus isolation test was used to determine the prevalence of calves viremic with BVDV on entry to the feedlot. An enzyme-linked immunosorbent assay (ELISA) which detects antibody against glycoprotein 53 of the BVDV was used on paired sera to determine the seroconversion risk during the first 96 d in the feedlot. A virus neutralization (VN) test for BVDV was conducted on a sub-sample of paired sera to measure agreement in determination of seroconversion risk with the ELISA. A polymerase chain reaction (PCR) test which detects BVDV was used to determine if cattle were acutely viremic when treated for disease. The estimated prevalence of persistently infected calves in this population was < 0.1%. The seroconversion risk for BVDV was 27% (236/864) according to the ELISA and it varied from 0 to 63% among the 20 pens sampled. According to the VN test, the seroconversion risk for BVDV was 40% (132/327) and it varied from 0 to 100% among the 11 pens tested. The agreement between the ELISA and VN tests in seroconversion risk to BVDV was very poor (kappa = 0.15 +/- 0.039 SE). The prevalence of acute viremia in calves treated at the feedlot hospital was low at 4% (6/149).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the interaction between VP8 of bovine rotavirus C486 and cellular components on MA-104 cells and erythrocytes.

Rotavirus VP8*, the N-terminal trypsin cleavage product of VP4, has been shown to bind to MA-104 cells and human O type erythrocytes. To examine whether bacterially expressed VP8* binds to cellular components of MA-104 cells, the VP8* (aa 1-247) was expressed in E. coli and radiolabelled with 35S-methionine. The radiolabelled rVP8* was immunoprecipitated with antiserum to bovine rotavirus C486 (BRV). The rVP8* was found to bind to MA-104 cells and its binding was competed by BRV. To study the interaction between VP8* and receptors of erythrocytes, hemagglutination (HA) and hemagglutination inhibition (HI) assays were carried out using solubilized rVP8*. rVP8* showed HA which could be inhibited by antiserum to BRV. This interaction was also inhibited by gangliosides, demonstrating a sialic acid dependent interaction. To study the contribution of the C-terminal region of VP8* to HA, a number of approaches were used. First, a peptide spanning aa 230-247 was synthesized and antisera was raised against the peptide to see whether it could inhibit HA of rVP8*. Second, a truncated form of VP8* (tVP8*: aa 1-229) was expressed to examine its hemagglutinating activity. Third, the dimerization of rVP8* and tVP8* was compared by Western-blotting following electrophoresis using native SDS-PAGE. The results indicated that antibody to aa 230-247 inhibits hemagglutination by preventing dimerization of VP8* which in turn allows the molecule to cause HA. To characterize the interaction between the HA domain and sialic acid receptors, erythrocytes were treated with sialidases of different specificities. Arthrobacter ureafaciens, Clostridium perfringens and alpha 2-8 linkage-specific neuraminidase destroyed the ability of sialic acid of erythrocytes to interact with rVP8*, indicating that bovine rotavirus C486 binding requires an alpha 2-8 linkage but acetylation of the sialic acid is not necessary.

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.

Performance, survival, necropsy, and virological findings from calves persistently infected with the bovine viral diarrhea virus originating from a single Saskatchewan beef herd.

Fifty-one calves from 652 cows and heifers that calved on a Saskatchewan ranch in 1992 were identified as persistently infected with bovine viral diarrhea virus (BVDV), based on virological and necropsy findings. Herd records suggested a further 20 calves that died between birth and weaning were probably also persistently infected. Subsequent to weaning, all surviving persistently infected calves were transferred to one pen in a 10,000 head commercial feedlot, to mimic normal management practice in western Canadian beef herds. On average, when compared with healthy, BVDV-negative herdmates, persistently infected calves were "poor doers" and had poor survivability, with only 4 persistently infected calves surviving to 1 year of age. There was no difference (P > 0.05) in survival between male and female persistently infected calves. The clinical, pathological, and virological findings from these persistently infected calves varied over time. The majority of persistently infected calves had gross pathological lesions at necropsy, consistent with mucosal disease. However, approximately 25% of the persistently infected calves had gross pneumonic lesions at necropsy, with no or only mild lesions of mucosal disease. A wide variety of other lesions were also noted in persistently infected calves at necropsy. Therefore, the possibility that BVDV-induced lesions can be misdiagnosed is very real. The results of this study indicate that persistent infection with BVDV should always be considered in calves with chronic ill thrift, chronic enteritis, or respiratory disease.

Investigation of an outbreak of mucosal disease in a beef cattle herd in southwestern Saskatchewan.

This study describes the epidemiological investigation of an outbreak of mucosal disease that occurred on a ranch in southwestern Saskatchewan. Over a six month period during the fall and winter of 1991-1992,in a herd of 515 beef cattle and 96 bison, 20 yearling cattle from a group of 105 housed in one feedlot pen died from mucosal disease. A further eight yearlings were slaughtered for salvage because they were at risk of dying from mucosal disease. Mucosal disease mortalities were the first observed evidence of fetal infections with bovine viral diarrhea virus in this herd. Animals that died from mucosal disease exhibited signs of ill thrift prior to death. Deaths from mucosal disease were confined to the progeny of one herd of beef cows. Following an outbreak of fetal infection with bovine viral diarrhea virus during 1989-1990, at least 28 (22%) of the 128 calves born from this herd of cows in the spring of 1990 were persistently infected with bovine viral diarrhea virus. However, only one calf born from this herd in 1991, and five calves born from all herds in 1992 were persistently infected. Of the five persistently infected calves born in 1992, three were born to persistently infected replacement heifers born in 1990. These heifers calved without assistance in 1992, but only one of their calves survived past three days of age, and it was persistently infected. In January 1992, 82% of the total herd had reciprocal antibody titers to bovine viral diarrhea virus of >/=1024 which suggested a high level of herd immunity to bovine viral diarrhea virus. Thus, following the outbreak of fetal infection with bovine viral diarrhea virus in 1989-1990, herd immunity to bovine viral diarrhea virus had developed rapidly in the breeding cows and heifers. Subsequently, in the next two years, there was a dramatic decline in the number of calves born persistently infected with bovine viral diarrhea virus.

Electroporation-based DNA transfer enhances gene expression and immune responses to DNA vaccines in cattle.

Despite the potential of DNA vaccines to induce strong, balanced immune responses in small experimental species, the immune responses to DNA immunization in larger species have generally been moderate and inconsistent. In this study, the TriGridtrade mark Delivery System (TDS), an electroporation-based DNA delivery platform, was evaluated for administration of DNA vaccines to calves. When compared to conventional intramuscular delivery, TDS-based delivery markedly and consistently enhanced gene expression from a plasmid encoding a reporter gene, secreted alkaline phosphatase, and improved cell-mediated and humoral immune responses to a plasmid encoding a model antigen, hepatitis B surface antigen. Importantly, the TDS-based procedure was well tolerated by the calves, which did not need to be anesthetized or sedated. These results suggest that the TDS is a useful delivery method for DNA vaccines in cattle.

DNA prime protein boost strategies protect cattle from bovine viral diarrhea virus type 2 challenge.

At present, infections with bovine viral diarrhea virus (BVDV) type 2 occur nearly as frequently as those with BVDV type 1, so development of vaccines that protect cattle from both type 1 and type 2 BVDV has become critical. In this study, we compared various DNA prime-protein boost vaccination strategies to protect cattle from challenge with BVDV-2 using the major protective antigen of BVDV, glycoprotein E2. Calves were immunized with a plasmid encoding either type 1 E2 (E2.1) or type 2 E2 (E2.2) or with both plasmids (E2.1+E2.2). This was followed by a heterologous boost with E2.1, E2.2 or E2.1 and E2.2 protein formulated with Emulsigen and a CpG oligodeoxynucleotide. Subsequently, the calves were challenged with BVDV-2 strain 1373. All vaccinated calves developed both humoral and cell-mediated immune responses, including virus-neutralizing antibodies and IFN-gamma-secreting cells in the peripheral blood. Depletion studies showed that CD4+ T cells were responsible for IFN-gamma production. Furthermore, the calves vaccinated with either the E2.2 or the E2.1+E2.2 vaccines were very well protected from challenge with BVDV-2, having little leukopenia and showing no weight loss or temperature response. In addition, the animals vaccinated with the E2.1 vaccine were partially protected, so there was a certain level of cross-protection. These data demonstrate that a vaccination strategy consisting of priming with E2.2 or E2.1+E2.2 DNA and boosting with E2.2 or E2.1+E2.2 protein fully protects cattle from BVDV-2 challenge.

Characterization of the structural proteins of hemorrhagic enteritis virus.

The structural proteins of hemorrhagic enteritis (HEV), a turkey adenovirus, were analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using polyspecific, monospecific and monoclonal antibodies for detection. In purified HEV preparations, eleven polypeptides with apparent molecular weights ranging from 96,000 to 9,500 (96k to 9.5k), were specifically recognized by convalescent turkey serum. Six of these polypeptides were further characterized by PAGE, Western blotting, ELISA, sucrose gradient centrifugation and electron microscopy. The 96k polypeptide was identified as the hexon polypeptide which is a monomer of the major outer capsid or hexon protein. The 51/52k and 29k polypeptides, identified as the penton base and fiber polypeptides respectively, were the components of the vertex or penton protein. The 57k polypeptide was identified as a homologue of the human adenovirus type 2 (Ad 2) IIIa protein with which it shares a common epitope. Two core proteins with molecular weights of 12.5 and 9.5k were present in purified HEV nucleoprotein cores. The proteins of two HEV isolates, one apathogenic (HEV-A) and one virulent (HEV-V), resembled each other in most respects. However, differences between HEV-A and HEV-V were found in electrophoretic migration of the penton base protein both under native and denatured conditions, and in the electrophoretic migration of the 43/44k polypeptide. Moreover, homologous antiserum against the fiber protein reacted stronger than heterologous antiserum in an ELISA. Single fibers were detected by electron microscopy attached to the penton base proteins of HEV virions and in isolated pentons. The feature of having single fibers is shared with the mammalian adenoviruses and the avian egg drop syndrome 1976 virus (EDS 76 V), but not with the fowl adenoviruses which have double fibers attached to their penton base proteins.

Protection of turkeys against haemorrhagic enteritis by monoclonal antibody and hexon immunization.

Virus-neutralizing monoclonal antibodies specific for the hexon of haemorrhagic enteritis virus (HEV), a turkey adenovirus, were examined for their ability to confer passive protection against haemorrhagic enteritis (HE) in turkeys. A high dose of antibody prevented clinical disease and reduced virus replication in experimentally infected birds. This suggests that virus neutralization might be an important mechanism for protection against HE. Subsequently, the use of the hexon protein as a subunit vaccine was investigated by immunizing birds with affinity-purified HEV hexon. The birds were tested for the appearance of hexon-specific antibodies in their sera, for protection from clinical disease, and prevention of virus replication after challenge with virulent HEV (HEV-V). Regardless of whether birds were immunized with native or denatured hexon, high ELISA antibody titres were produced to each immunogen. A virus-neutralizing antibody response was induced by immunization with the native hexon but not by immunization with the denatured protein. All turkeys twice immunized with a dose of at least 1 microgram, and four out of five birds immunized with two doses of 0.3 micrograms of purified native hexon, were protected against virus-induced disease and virus replication. In contrast, birds inoculated with denatured hexon were not protected. These results demonstrate the importance of the native (trimeric) structure of the hexon protein for eliciting a protective immune response. The impact of these results on the development of a vaccine for HE in turkeys produced by recombinant DNA technology is discussed.

Characterization of the haemorrhagic enteritis virus genome and the sequence of the putative penton base and core protein genes.

Haemorrhagic enteritis virus (HEV) is a member of a genetically ill-defined group within the genus Aviadenovirus which causes significant clinical disease in gallinaceous fowl. Using DNA obtained from a low virulence isolate of HEV passed in turkeys, we developed a genomic restriction map and estimated an apparent genomic length of 25.5 kb. No evidence for extensive DNA hybridization was found between the HEV genome and either the hexon or penton base genes of human adenovirus 2 (HAdV-2) and fowl adenovirus 10 (FAdV-10). The HEV penton base gene was identified by PCR using primers based on conserved adenoviral DNA sequences. The penton base gene was expressed in Escherichia coli as a fusion protein and detected by anti-HEV serum in both colony and denaturing gel immunoblots. DNA sequencing revealed a putative penton base ORF with a predicted amino acid sequence showing approximately 39.0%, 53.0% and 44.2% similarity with the penton base of HAdV-2, human adenovirus 40 (HAdV-40) and FAdV-10, respectively. The penton base gene was located at 43.3-48.6 m.u. on the HEV genome and had a remarkably low G+C content (33.8%). DNA sequencing also revealed ORFs for putative core proteins resembling pVII, p-mu and a partial ORF similar to pVI (hexon-associated protein) of HAdV-2 and HAdV-40. The results support the claim that HEV represents a distinct group of viruses within the genus Aviadenovirus.

Topographical analysis of bovine herpesvirus type-1 glycoproteins: use of monoclonal antibodies to identify and characterize functional epitopes.

Monoclonal antibodies, specific for two of the major bovine herpesvirus type-1 (BHV-1) glycoproteins, GVP 3/9 and GVP 6/11a/16, have been previously produced and characterized. The reactivity of each monoclonal antibody was determined by enzyme-linked immunosorbent assay (ELISA), the ability to neutralize viral infectivity, and the capacity to mediate complement-dependent (AbC') lysis of virus-infected cells. The topography of epitopes on GVP 3/9 and GVP 6/11a/16 was analyzed, using selected monoclonal antibodies in a competitive antibody binding assay (CBA). Nine epitopes were identified on GVP 3/9. Comparison of the biological activities and epitope specificities of the monoclonal antibodies against GVP 3/9 showed that one epitope was involved in virus neutralization, whereas six epitopes mediated AbC' lysis of the virus-infected cell. Of the seven epitopes identified on GVP 6/11a/16, six were involved in neutralization and three participated in AbC' lysis. In some cases, partial competition between monoclonal antibodies was observed, indicating that they were directed against overlapping or closely adjacent epitopes. In other instances, asymmetrical competition between monoclonal antibodies suggested conformational changes in the glycoprotein molecule induced by antibody binding. Mixtures of two monoclonal antibodies with different epitope specificities resulted in higher neutralizing activity than either antibody alone, suggesting that multiple monoclonal antibodies can exert a synergistic effect on virus neutralization.

Protection of cattle from BHV-1 infection by immunization with recombinant glycoprotein gIV.

High levels of recombinant bovine herpesvirus-1 (BHV-1) glycoprotein IV were produced in baculovirus, adenovirus, vaccinia virus and Escherichia coli expression systems. The different recombinant forms as well as authentic gIV were injected intramuscularly into seronegative calves. With the exception of E. coli-produced gIV, all forms of gIV induced high levels of neutralizing antibodies both in the serum and in the nasal superficial mucosa. Animals immunized with gIV produced in insect or mammalian cells were completely protected from infection with BHV-1, as demonstrated by the absence of temperature responses, clinical signs or detectable virus in the nasal secretions after challenge exposure. The E. coli-derived gIV induced partial protection from clinical disease, even though it was not glycosylated and did not induce appreciable levels of neutralizing antibodies. This study demonstrated that all forms of glycosylated gIV, whether authentic or recombinant, confer protection from BHV-1 infection and thus may be useful as an effective subunit vaccine.

Role of N-linked glycans in antigenicity, processing, and cell surface expression of bovine herpesvirus 1 glycoprotein gIV.

Glycoprotein gIV, a structural component of bovine herpesvirus type 1, stimulates high titers of virus-neutralizing antibody. The protein contains three potential sites for the addition of N-linked carbohydrates. Three mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Thr/Ser to Ser-X-Thr/Ser. A fourth mutant was altered at two sites. The altered forms of the gIV gene were cloned into a vaccinia virus transfer vector to generate recombinant vaccinia viruses expressing mutant proteins. Analysis of these mutants revealed that only two (residues 41 and 102) of the three (residues 41, 102, and 411) potential sites for the addition of N-linked glycans are actually utilized. Absence of glycans at residue 41 (gN1) showed no significant effect on the conformation of the protein or induction of a serum neutralizing antibody response. However, mutant proteins lacking glycans at residue 102 (gN2) or residues 41 and 102 (gN1N2) showed altered reactivity with conformation-dependent gIV-specific monoclonal antibodies. These mutants also induced significantly lower serum neutralizing antibody responses than wild-type gIV. Nonetheless, each of the mutant proteins were modified by the addition of O-glycans and transported to the cell surface. Our results demonstrate that absence of N-linked glycans at one (residue 102) or both (residues 41 and 102) utilized N-linked glycosylation sites alters the conformation but does not prevent processing and transport of gIV to the cell surface.

A subunit gIV vaccine, produced by transfected mammalian cells in culture, induces mucosal immunity against bovine herpesvirus-1 in cattle.

A truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein IV (tgIV) was produced in a novel, non-destructive expression system based upon regulation of gene expression by the bovine heat-shock protein 70A (hsp70) gene promoter in Madin Darby bovine kidney (MDBK) cells. In this system, up to 20 micrograms ml-1 of secreted tgIV, which is equivalent to the yield from 4 x 10(6) cells, was produced daily over a period of up to 18 days. Different doses of tgIV were injected intramuscularly into seronegative calves. Virus-neutralizing antibodies were induced by all doses of tgIV, both in the serum and in the nasal superficial mucosa. However, the low dose (2.3 micrograms) induced significantly (p < 0.05) lower antibody titres than the medium (7 micrograms) and high (21 micrograms) doses. The medium and high doses of tgIV conferred protection from BHV-1 infection, as demonstrated by a significant (p < 0.05) reduction in clinical signs of respiratory disease and virus shedding in the nasal secretions postchallenge. However, the 2.3 micrograms group, although partially protected, was not significantly (p > 0.05) different from the placebo group. This study demonstrated the potential of an intramuscularly administered tgIV subunit vaccine to induce mucosal immunity to BHV-1 using an economic protein production system and an acceptable vaccine formulation. In addition, a strong correlation was observed between neutralizing antibodies in the serum and nasal superficial mucosa, virus shedding and clinical disease. Thus, serum neutralizing antibody levels in tgIV-immunized animals may be a good prognosticator of protection from BHV-1 infection and disease.

Functional characterization of bovine parainfluenza virus type 3 hemagglutinin-neuraminidase and fusion proteins expressed by adenovirus recombinants.

We constructed replication-competent human adenovirus type 5 (HAd5) recombinants (HAd5-HN and HAd5-F) containing the bovine parainfluenza virus type 3 (BPIV3) hemagglutinin-neuraminidase (HN) or fusion (F) gene under the control of the simian virus 40 (SV40) regulatory sequences. These genes were inserted in the early region 3 (E3) of the HAd5 genome in the E3 parallel orientation. Expression of HN or F in HAd5-HN- or HAd5-F-infected cell extracts, respectively, was observed by immunoprecipitation using a BPIV3-specific polyclonal antiserum. Our results suggest that HN and F expressed by HAd5 recombinants were functionally similar to the native HN and F expressed in BPIV3-infected cells.