Failure of foetal protection after vaccination against an experimental infection with bovine virus diarrhea virus.

A protocol is described to measure the protection of the bovine fetus against an experimental bovine virus diarrhea virus (BVDV) infection after vaccination. Two inactivated experimental vaccines were applied twice with a 3 week interval. A mixture of three different Dutch field strains was used as challenge on mainly the 82nd day of gestation to vaccinated and unvaccinated control animals. The challenge was applied 5 months after completion of the two-fold vaccinations. All calves born from unvaccinated control animals were persistently infected. The calves born from dams vaccinated with the two different inactivated BVDV vaccines were persistently infected in 78 and 60%, respectively.

A comparative study of serological tests for use in the bovine herpesvirus 1 eradication programme in The Netherlands.

We compared a gB-ELISA, a gE-ELISA and a Danish test system (consisting of a blocking and an indirect ELISA) for their specificity and sensitivity to detect antibodies against BHV1. The Danish test system showed the highest sensitivity and the gE-ELISA the lowest; the gB-ELISA showed an intermediate sensitivity. If the doubtful zone (25-50% blocking) of the gB-ELISA was considered as positive (gB-ELISA+), the sensitivity almost reached that of the Danish test system. The specificity of all tests appeared to be very high, 99.7, 96.7, 100, 99.7% for the gB-ELISA, gB-ELISA+, gE-ELISA and the Danish test system, respectively. Seroconversion was detected in the gE-ELISA up to 3 weeks later than in the gB-ELISA and the Danish test system. It is concluded that the combination of a gB-ELISA (for screening) and the Danish test (for confirmation) system used in the BHV1 eradication programme in the Netherlands, provides for very high sensitivity (> 99.0%) (Kramps et al., 1994) and a very high specificity (> 99.9%).

Detection of specific IgM antibodies to infectious bronchitis virus by an antibody-capture ELISA.

The results of a new antibody-capture ELISA (alpha-IgM-IBV ELISA), specific for IgM directed against Infectious Bronchitis Virus (IBV) show that this assay is a useful tool for diagnosing IBV infections. The data include individual results of the alpha-IgM-IBV ELISA in sequential SPF chicken sera after vaccination with H120 and challenge with M41, the specificity is based on results of 499 SPF sera, and the sensitivity on sera from experimentally vaccinated and challenged birds. Also reported are ELISA results on 168 field sera originating from seven broiler flocks (24 samples per flock) collected during the acute phase of an IBV infection. The alpha-IBV-IgM ELISA results obtained with field sera from broiler flocks infected by Massachusetts, D207, D212 or an unidentified serotype of IBV indicated that the ELISA, in which only M41-strain was used as antigen, was able to detect IgM responses to IBV serotypes other than Massachusetts. The specificity of the alpha-IBV-IgM ELISA was 99%, the sensitivity based on the experimental vaccination was 83 to 100%, depending on the day post vaccination. The sensitivity, based on the experimental challenge, was 83%, while the sensitivity, based on 168 field sera, was 93.5%. The IgM responses were rapid and transient and therefore indicative for acute IBV infections.

Comparative study on four enzyme-linked immunosorbent assays and a cocultivation assay for the detection of antigens associated with the bovine viral diarrhoea virus in persistently infected cattle.

Four commercially available ELISAs for detection of antigens associated with the bovine viral diarrhoea virus in persistently infected cattle have been compared. The tests are equally specific (100%) and the sensitivity of three ELISAs is comparable with that of a conventional cocultivation assay. Performing ELISA on samples from young animals that received colostrum may yield false negative results because of interference of maternal antibodies in the tests. It is concluded that ELISA can be a valuable tool in eradications programs when large numbers of cattle are to be monitored.

Virulence and genotype of a bovine herpesvirus 1 isolate from semen of a subclinically infected bull.

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.

[Serological study of the occurrence of L. hardjo in sheep in The Netherlands]. / Een serologisch onderzoek naar het voorkomen van L. hardjo bij schapen in Nederland.

Sheep (3918) from 137 farms in the regions of North-, West- and Mid-Netherlands and Gelderland were serologically investigated for the presence of antibodies against Leptospira hardjo. Antibodies were detected in 3.3% of the sheep. There were large regional differences with respect to both the percentage of positive sheep and the percentage of positive flocks. All sera from sheep in Gelderland were negative. In West- en Mid-Netherlands there were 0.9 and 6.5% positive sera, respectively, and 19.4 and 32.7% positive flocks. The percentage of positive sheep per positive flocks varied from 1 tot 51.6. Serological positive sheep were from farms with and without cattle. On farms with cattle, there was no clear relationship between serologically sheep and the presence of hardjo antibodies in cattle. It thus seems plausible that sheep can be infected with hardjo independently of cattle.

A subclinical infection of bulls with bovine herpesvirus type 1 at an artificial insemination centre.

A subclinical bovine herpesvirus type 1 (BHV1) infection affected bulls at an artificial insemination centre for at least six months. The virus was detected in semen samples from 43 out of 116 bulls examined; 27 of them shed virus during one consecutive period of up to 10 days and 16 shed the virus intermittently. The virus titres ranged between 10 and 100,000 TCID50/ml. Vaccinated bulls tended to excrete the virus less frequently than unvaccinated bulls. Approximately 50 per cent of the bulls that had an increase in neutralising antibody titre to BHV1 shed the virus in their semen.

Immunity to human and bovine respiratory syncytial virus.

Human and bovine respiratory syncytial viruses resemble each other closely. During annual winter outbreaks, they cause similar respiratory tract disease in infants and calves. The disease is most severe in children and calves between 1 and 3 months old, when maternal antibodies against the virus are usually present. Reinfections, which are common, are accompanied by progressively milder illnesses in children, but are symptomless in calves. Because maternal antibodies suppress serum and mucosal antibody responses of all isotypes, the development of a vaccine that is effective in young children and calves with high levels of maternal antibodies has been severely hampered. Although virus administered intranasally to young calves with maternal antibodies does not evoke antibody responses, it can prime these calves for a protective memory response upon reinfection. Protection appears to be associated with the capacity to mount a mucosal memory IgA response. There are several indications that one or more immunopathologic mechanisms contribute to the disease. An Arthus reaction (type III) may have a role in the pathogenesis, because activated complement may cause most of the pathologic lesions, including edema and emphysema in uninfected parts of the lung. Lungs from calves with severe or fatal disease have depositions of complement component C3 and a low histamine content. The most immunogenic and protective antigen of the virus is the fusion (F) glycoprotein, which evokes a strong antibody response and is a target for cytotoxic T cells. On the F protein, epitopes that induce neutralizing and non-neutralizing antibodies, both of which may enhance complement activation, were identified. Immunity to the F protein may have beneficial and harmful effects.

A severe outbreak of respiratory tract disease associated with bovine respiratory syncytial virus probably enhanced by vaccination with modified live vaccine.

A severe outbreak of respiratory tract disease associated with bovine respiratory syncytial virus (BRSV) on a large beef-fattening farm is described. The outbreak started two days after five- to seven-month-old calves were vaccinated with a modified live BRSV vaccine. The disease ran a very severe course among five- to seven-month-old vaccinated calves, but disease was absent in eight-month-old an older non-vaccinated calves. The presence of IgM antibodies in sera of non-vaccinated calves indicated that BRSV was spreading on the farm between two to 15 days before the day of vaccination. The data indicate that vaccination with modified live vaccine during the course of a natural infection may enhance the severity of disease. The possible pathogenesis of the disease is discussed.

Priming for local and systemic antibody memory responses to bovine respiratory syncytial virus: effect of amount of virus, virus replication, route of administration and maternal antibodies.

We studied the conditions under which calves can be primed for mucosal and serum antibody memory responses against bovine respiratory syncytial virus (BRSV), and the relationship between such responses and protection against the virus. Calves were primed via the respiratory tract with a low or high amount of live virus, with killed virus, or intramuscularly with live virus. Calves were challenged via the respiratory tract. Priming with live virus via the respiratory tract induced primary antibody responses in serum and on the mucosae, which were identical after the low and the high amount of virus. These responses were suppressed by maternal antibodies. Intramuscular priming of seronegative calves induced serum IgG1 and sometimes serum IgM and IgG2 responses, but no responses were detected on the mucosae. Sera of calves primed by the intramuscular or the respiratory route recognized the same viral proteins. No responses were observed after priming with killed virus, or after intramuscular priming of calves with maternal antibodies. After challenge, mucosal and serum antibody memory responses developed in calves that had been primed via the respiratory tract with live virus, whether they had maternal antibodies or not. One colostrum-fed calf showed a mucosal memory response, although serum responses were still suppressed by maternal antibodies. None of the calves thus primed shed virus after challenge. Intramuscular priming also primed for mucosal and serum memory responses after challenge, which however started perhaps slightly later and were not associated with protection against virus shedding. Priming with killed virus, or with live virus intramuscularly in the presence of maternal antibodies proved least effective in inducing memory and protection against virus shedding. Thus, protection against virus shedding was afforded by priming with live virus via the respiratory tract, both in calves with an without maternal antibodies. Protection was associated with a strong and rapid mucosal antibody memory response, but the reverse was not necessarily true. Protection against virus excretion had no relationship to titers of serum neutralizing or serum IgG1 or nasal IgA antibodies at the time of challenge.

Development of a neutralising antibody response to an inoculated cytopathic strain of bovine virus diarrhoea virus.

Fourteen clinically healthy cattle that were persistently infected with non-cytopathic bovine virus diarrhoea virus (BVDV) and three BVDV-free cattle were inoculated with one of three cytopathic BVDV strains. Mucosal disease developed in 12 of the viraemic cattle, resulting in a moribund condition 17 to 99 days after inoculation. Two of the viraemic cattle remained clinically healthy until the end of the experiment, 14 months after inoculation. The BVDV-free cattle did not develop clinical signs after inoculation. From each cow with mucosal disease a noncytopathic and a cytopathic BVDV strain were isolated from tissue specimens collected post mortem. All the cattle developed moderate to high levels of neutralising antibodies against the cytopathic BVDV strain with which they were inoculated. The antibodies from 10 of the 12 cattle with mucosal disease did not react with the cytopathic BVDV strains isolated post mortem, and antibodies from none of them reacted with the non-cytopathic BVDV isolates. Antibody responses to the inoculated BVDV strains developed earlier in the viraemic cattle than in the BVDV-free cattle.

Activation of complement by bovine respiratory syncytial virus-infected cells.

Because complement activation is probably involved in the pathogenesis of as well as in recovery from the disease induced by bovine respiratory syncytial virus (BRSV), we studied the activation of complement by BRSV-infected cells in vitro in a homologous system. Binding of C3 on the surface of infected cells was measured in a biotin-streptavidin amplified ELISA, and complement-mediated lysis was measured in a 51Cr release assay. Without antibody, infected cells activated and bound more C3 than uninfected cells. C3 activation that occurred in the absence of antibody was largely mediated by the classical pathway and induced lysis inefficiently. BRSV-specific antibody enhanced complement activation as measured by both C3 ELISA and cytotoxicity assay. In the presence of antibody, C3 activation was largely dependent on the alternative pathway and efficiently induced lysis. Both IgG1 and IgM antibodies enhanced C3 activation, but IgG2 and IgA did not enhance C3 activation in our experiments. Preincubating cells with IgA or IgG2 did not inhibit C3 activation enhanced by IgG1 or IgM. Murine monoclonal IgG1 antibodies against epitopes on the Fusion protein of the virus also enhanced C3 binding, but differed in their capacity to induce complement-mediated lysis.

Pathogenesis of naturally acquired bovine respiratory syncytial virus infection in calves: evidence for the involvement of complement and mast cell mediators.

Indicators of immune-mediated disease were studied in calves with severe natural bovine respiratory syncytial virus infection. Although antigen and antibody were detected concurrently in most calves, immune complexes were not detected by use of immunofluorescence, ELISA, and binding of the 1q component of complement. Complement component C3, however, was observed by immunofluorescence in the cranioventral, virus-infected portion of the lungs of 19 of 25 calves. Reductions in the amount of histamine and in the numbers of mast cells and mast cell granules in the virus-positive cranioventral and virus-negative caudodorsal portions of the lungs, indicated activation of mast cells and liberation of their granule contents. On the basis of these and previous findings, a model for the pathogenesis of bovine respiratory syncytial virus-induced disease was proposed.

Characterization of the F41 fimbrial antigen of enterotoxigenic Escherichia coli by using monoclonal antibodies.

We produced monoclonal antibodies (MAbs) from 23 different murine hybridoma cell lines against the F41 fimbrial antigen of bovine and porcine enterotoxigenic Escherichia coli. Cell lines were created by fusing myeloma cells and spleen cells of mice that were immunized with either purified F41 or with Formalin-killed whole cells. The specificity of the MAbs to the F41 antigen was proven by enzyme-linked immunosorbent assays (ELISAs) and radioimmunoprecipitation tests. Epitope analysis with a competition ELISA revealed that the 23 MAbs recognized at least five epitopes. These results were corroborated by those of immunodiffusion tests, in which all possible combinations of two MAbs were tested against ultrasonically disintegrated F41 antigen. In a double-antibody sandwich ELISA, all peroxidase-conjugated MAbs bound to the F41 antigen of all 182 bacterial strains that were tested. Apparently, the epitopes recognized by the MAbs are highly conserved. Immunoelectron microscopy revealed that the MAbs were directed to fimbrial structures 3 to 4 nm in diameter and that the epitopes were equally distributed along the fimbriae. Consequences for the replacement of polyclonal antisera by MAbs in diagnostic tests are discussed. The results of the radioimmunoprecipitation assay suggested that F41 fimbriae are composed of a single repeating 29,000-dalton protein subunit; however, we could not exclude the possibility of the existence of minor fimbrial components.

Analysis of the antibody response to bovine respiratory syncytial virus proteins in calves.

The antibody response in calves to natural infections with bovine respiratory syncytial virus (BRSV) was analysed by radioimmunoprecipitation assays. Antibodies to virus proteins of Mr 200K (L), 87K (G), 46K (F1), 41K (N), 35K (P), 28K and 24K (F2), 27K (M), 22K and less than 14K could be identified. Recovery of 6- to 7-month-old calves from severe BRSV-associated disease was accompanied by the development of an antibody response to the virus, which was directed mainly o the F and N proteins. Calves of 2 to 3 weeks of age possessed moderate levels of maternal antibodies to BRSV particularly directed to the F and N proteins but became seriously ill after infection. The antibody response in these calves was severely suppressed. In the sera of 4- to 9-month-old calves that died in the course of infection, high antibody levels to the virus were found, which were directed at least to the F and N proteins. The presence or development of antibodies to the F and N proteins appears insufficient for protection against or recovery from BRSV infections.

An enzyme-linked immunosorbent assay for detection of antibodies to porcine parvovirus.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of antibodies to porcine parvovirus (PPV). Antisera to PPV were raised in pigs, for which PPV grown on PK15 cells was used for primary intranasal inoculations, and PPV cultured on autologous kidney cells for booster immunisations. A competition ELISA was developed, based on the principle of a double antibody sandwich assay, using immunoglobulin fractions prepared from these sera. The ELISA was compared with a haemagglutination inhibition (HI) test. The tests were equally sensitive for detecting antibodies early after infection and for detecting a significant increase in antibody titre between paired sera. A high correlation was found between antibody titres of field sera measured by the two tests (r = 0.91). We conclude that ELISA is preferable to the HI test, because it is labour-saving and can be standardised better and automated.

Epidemiological study of bovine respiratory syncytial virus infections in calves: influence of maternal antibodies on the outcome of disease.

A prospective epidemiological survey on bovine respiratory syncytial virus (BRSV) infections in calves was carried out on 21 dairy farms during one BRSV epidemic season. Special attention was paid to the role of maternal antibodies. On 15 farms the spread of the virus was demonstrated during the investigation period and on eight farms this was accompanied by an outbreak of acute respiratory disease. Disease seldom occurred in calves younger than two weeks old and the most severe disease was observed in calves from one to three months old. Although maternal antibodies did not effectively prevent the disease, both the incidence and severity of disease were inversely related to the level of specific maternal antibodies. Two serodiagnostic techniques were compared. In calves older than three months from herds with disease outbreaks associated with bovine respiratory syncytial virus the diagnosis was established in 80 per cent of the animals by an increase in IgG titre against BRSV and in 77 per cent by the detection of BRSV specific IgM. In comparison, only 10 per cent of the calves younger than three months were positive by IgG serodiagnosis, and 51 per cent by IgM serodiagnosis. On farms where the spread of the virus was accompanied by an outbreak of clinical disease more calves were present, a higher proportion of the calves was younger than three months, and calves of all ages were more often housed together.

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus.

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus (BRSV) are described. BRSV-specific IgG1 and IgG2 were determined in indirect double antibody sandwich assays. For IgA and IgM antibody capture assays were used. The isotype specificity of the assays was confirmed by the observation that samples with a high titre of BRSV-specific antibodies of particular isotype were negative in the assays for the other isotypes and vice versa. Comparison of the results obtained in the ELISAs and in the virus neutralisation test showed that acute phase antibodies were more efficiently detected in the latter. It also showed that the presence of BRSV-specific IgA was not correlated with neutralising activity in vitro. The serum antibody response of BRSV-infected seronegative calves from the field consisted of a nearly simultaneous increase of IgM, IgA and IgG1-antibodies in the acute phase of the disease, while the IgG2-response followed at various intervals thereafter. In young animals with maternal antibodies a different pattern was found. There was no increase in IgG1 and IgG2, but six of eight animals showed a weak IgM response and two of these six calves also showed a weak and short lasting IgA response. Because maternal antibodies are insufficiently effective in protecting calves against BRSV, the presence of such antibodies at mucosal surfaces was investigated. Maternal immunity was found to be restricted to IgG1 antibodies in serum. This agrees with the failure of maternal antibodies to protect mucosal surfaces against BRSV infection.

Immunoglobulin M-specific enzyme-linked immunosorbent assay for serodiagnosis of bovine respiratory syncytial virus infections.

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin (Ig) M antibodies to bovine respiratory syncytial virus (BRSV) in cattle was developed. Monoclonal antibody to bovine IgM was used as the catching antibody. The IgM-ELISA was used, as well as a BRSV-specific IgG ELISA to determine the kinetics of IgM and IgG antibody responses to BRSV infections in cattle. High IgM and IgG antibody titers developed after naturally occurring or induced BRSV infection of calves (6 to 7 months old). Induced infection resulted in an IgM response that was first detectable at postinoculation day (PID) 11 reached a maximum at PID 13, and became undetectable again about PID 28. An IgG response also was detected by PID 11. However, a maximum response was not reached before PID 23, and titers remained high (until PID 80). In naturally occurring infection, IgM and IgG responses in calves were observed in the acute phase of epizootics of respiratory tract disease. Patterns of IgM and IgG response curves were similar to those observed in experimentally infected calves. The involvement of BRSV in an epizootic of respiratory tract disease in 8 calves (2 to 3 weeks old) was demonstrated by the detection of BRSV in several lung lavage samples. All calves had existing IgG antibodies to BRSV which were interpreted to be maternally derived. None of the calves responded with an increase in IgG antibody titer. However, a weak but distinct BRSV IgM antibody response occurred in 6 calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Local and systemic antibody response to bovine respiratory syncytial virus infection and reinfection in calves with and without maternal antibodies.

Enzyme-linked immunosorbent assays for the detection of immunoglobulin M (IgM), IgA, IgG1, and IgG2 antibodies against bovine respiratory syncytial virus (BRSV) were used to measure antibody responses of calves after experimental or natural infection with BRSV. Serially collected sera, lung lavage samples, nasal and eye secretions, and feces were tested for the presence of these antibodies. Lung lavage fluids and nasal secretions were further examined for the presence of virus. After experimental infection of 3- to 4-week-old, colostrum-deprived (seronegative) calves, the virus was detected from days 3 to 8 post-initial inoculation day (PID). An immune response was first detected 8 to 10 days PID, when BRSV-specific IgM and IgA appeared nearly simultaneously in serum, secretions, and feces. BRSV-specific IgG1 appeared only in serum on days 13 to 17 PID, and IgG2 was first detected in sera from 1 to 3 months PID. Specific IgM and IgA were detectable in the different samples for various periods. In the respiratory and eye secretions, IgA usually remained detectable for long periods, that is, for up to 3.5 months or longer. In lung lavage samples, BRSV-specific IgG1 was only incidentally demonstrated and appeared to be blood derived. The immune response of a 5-month-old calf strongly resembled that of the 3- to 4-week-old calves (feces excepted), indicating that an age effect on the immune response to BRSV is unlikely. After experimental infection of colostrum-fed, seropositive calves, both local and systemic antibody responses were largely or totally suppressed. The degree of suppression seemed to be related to the level of preinoculation virus-specific serum IgG1. Of all isotypes, IgM was least affected. Colostrum-fed animals shed virus in about equal amounts and for the same length of time as colostrum-deprived calves. Clinical signs were mild in both groups. After reinfection, no virus shedding was detected in either colostrum-deprived or colostrum-fed calves. In both groups, a secondary immune response developed, characterized by strong and rapid (from about day 6 PID) mucosal and systemic IgA responses, but reaching higher titers in colostrum-deprived calves. Also, strong mucosal, but not serum, IgM responses were observed, which, however, did not develop faster than those observed after primary infection. Naturally infected calves, showing severe signs of respiratory disease, had various levels of, most likely, maternally derived antibodies on the first day of illness. Mucosal and systemic antibody responses of various heights and durations were observed, but in general these responses were stronger than those observed after experimental infection. The results point to an important role for local IgA, rather for serum IgG1, in the protection against BRSV infection. The capacity to mount a local memory IgA response seems especially important. Priming for such a mucosal memory response is possible even when the primary immune response is severely suppressed because of the presence of material antibodies.