J. A. Kramps

Respiratory syncytial virus infections in human beings and in cattle

Respiratory syncytial virus (RSV) causes yearly outbreaks of respiratory disease in human beings and cattle all over the world. Most severe human respiratory syncytial virus (HRSV)-associated disease is observed in children less than 1 year of age while most severe bovine respiratory syncytial virus (BRSV)-associated disease is observed in calves less than 6 months of age. Two subgroups of HRSV have been identified. The existence of two subgroups of BRSV has been repeatedly suggested but is not yet well established. BRSV and HRSV are closely related antigenically but antigenic differences have been observed. Seasonal periodicity of RSV infection is usual with highest incidences in autumn and winter. Stress such as caused by movement, crowding and temperature changes are considered to play a role in bovine outbreaks. Human beings and cattle are the natural hosts of HRSV and BRSV, respectively. Primarily infected individuals are the most important source of RSV during outbreaks. The role of other species in the spread of HRSV and BRSV is unknown. Protective efficacy of maternally derived antibodies is considered to be incomplete. Such antibodies do not reduce shedding of virus after HRSV and BRSV infection. RSV is often transmitted by contact with nasal secretions but may also be transmitted by aerosols. Seroprevalence of HRSV and BRSV among adult human beings and cattle is over 70% and is always higher than it is among younger individuals. Both human beings and cattle of all ages may be reinfected with RSV. During BRSV reinfections, signs of respiratory tract disease and shedding of virus are seldom observed whereas these are common during HRSV reinfections. Persistent HRSV and BRSV infections in human beings and cattle have been suggested but have not so far been reported.

Dynamics of bovine respiratory syncytial virus infections: a longitudinal epidemiological study in dairy herds

SummaryTo study the epidemiology of respiratory syncytial virus (RSV) infections during the year, the incidences of primary infections and reinfections were monitored by titrating antibodies to bovine RSV (BRSV) in cattle above 2 months of age in 6 dairy herds in the Netherlands. From August 1990 until September 1991, 884 cattle were sampled at one-month intervals. A total of 155 cattle, most under two years of age, had a primary antibody response. Antibody rises were found in 259 cattle of all ages. The highest incidences of BRSV infections were found in one period either in autumn or winter. In other seasons, primary infections were rare, whereas reinfections were not uncommon. In 5 out of the 6 herds, two seronegative sentinel calves were introduced at the end of the winter and none developed specific antibodies before the next winter. The observations strongly suggest that, in spite of regular reinfections, BRSV circulates during spring or summer at a very low level or not at all. Persistent BRSV infection in a number of cows might be a means for the virus to survive during summer, but a steady rate of reinfection of seropositive cows throughout the year at a low level might also maintain a reservoir of infectious virus. This study adds to the knowledge of frequency and timings of primary infections and reinfections of BRSV and it might contribute to the study of these issues of human RSV.

Immunization of cattle with a BHV1 vector vaccine or a DNA vaccine both coding for the G protein of BRSV

A gE-negative bovine herpesvirus 1 (BHV1) vector vaccine carrying a gene coding for the G protein of bovine respiratory syncytial virus (BRSV) (BHV1/BRSV-G) induced the same high degree of protection in calves against BRSV infection and BHV1 infection as a multivalent commercial vaccine. A DNA plasmid vaccine, carrying the same gene as the BHV1/BRSV-G vaccine, significantly reduced BRSV shedding after BRSV infection compared with that in control calves, but less well than the BHV1/BRSV-G vaccine. Flow cytometric analysis showed a significant relative increase of gamma/delta+ T cells in peripheral blood after BRSV challenge-infection of the calves of the control group but not in the vaccinated groups. These results indicate that the G protein of BRSV can induce significant protection against BRSV infection in cattle, and that the BHV1/BRSV-G vaccine protects effectively against a subsequent BRSV and BHV1 infection.

Type-specific serologic diagnosis of respiratory syncytial virus infection, based on a synthetic peptide of the attachment protein G

Peptides deduced from the central hydrophobic region (residues 158-189) of the G protein of bovine and ovine respiratory syncytial virus (RSV) and of human RSV subtypes A and B were synthesized. These peptides were used to develop ELISAs to measure specifically antibodies against these types and subtypes of RSV. We have evaluated the bovine RSV-G peptide in both an indirect ELISA and in a blocking ELISA. Specificity and sensitivity, relative to a routine diagnostic ELISA that detects antibodies against the RSV F-protein in bovine sera, were 98% and 92% respectively for the indirect peptide-based ELISA, and 98% and 98% for the blocking peptide-based ELISA. In paired serum samples, rises in antibody titer were detected more frequently with the indirect peptide-based ELISA than with the routine F-ELISA. Furthermore, the peptide-based G-ELISAs were able to differentiate between antibodies against BRSV and HRSV, and between those against BRSV and ORSV. In addition, the indirect peptide-based ELISA was selective for HRSV subtype A and B antibodies. This study shows that peptides, corresponding to the central hydrophobic region of the attachment protein G of several RSVs, can be used successfully as antigens in highly specific and sensitive immunoassays.

Experimental reproduction of respiratory disease in calves with non‐cell‐culture‐passaged bovine respiratory syncytial virus

To reproduce experimentally clinical bovine respiratory syncytial virus (BRSV) infections in cattle, we isolated BRSV from a calf in the field that suffered from acute respiratory disease. Cell culture passage of the virus was avoided to prevent any modification of the biological properties of the virus. The isolated BRSV was passaged in specific-pathogen-free (SPF) calves. Lung lavage fluids of these calves, which contained at least 10(3) TCID50/ml BRSV and which were found to be free of other known respiratory pathogens, were collected and pooled for experimental infection. To reproduce a clinical BRSV infection, two groups of six SPF calves were inoculated intranasally with 2 ml of 10(3.9) TCID50/ml BRSV of the obtained virus stock. Another five calves, which were persistently infected with bovine virus diarrhoea virus (BVDV), were given the same inoculum. One group of six calves served as mock-infected controls. Clinical signs were closely monitored from 1 week before until 16 days after inoculation. Reproducible clinical signs consisting of significantly (p < 0.05) increased respiratory rates and elevated body temperatures were recorded but not in all BRSV-inoculated calves. Although clinical signs were induced by experimental infection with non-cell-culture-passaged BRSV, the respiratory signs were not as serious as in the most severe cases in the field.

A European inter-laboratory trial to evaluate the reliability of serological diagnosis of bovine herpesvirus 1 infections

The detection of cattle latently infected with bovine herpesvirus 1 (BHV1) is of importance in control programs and in international trade activities. Therefore, tests to detect specific antibodies in serum must be highly sensitive. To evaluate the reliability of serological diagnosis of BHV1 infections in Europe, seventeen laboratories in 15 European countries were asked to determine BHV1-specific antibodies in a panel of bovine serum samples using the serological tests available in their laboratory. Laboratory tests included virus neutralisation tests (1, 2 and 24 h), indirect immunofluorescence tests and ELISAs. The serum panel consisted of 12 duplicate lyophilised samples which were randomly coded from 1 to 24 and included negative, weak- and strong-positive samples as well as international reference sera. Virus neutralisation tests and ELISAs showed a high specificity. All participants using neutralisation tests (n = 13) scored the negative samples correctly. Twenty three of 25 ELISAs showed 100% specificity. A serum sample obtained at day 7 after infection was scored as negative by all tests except one home-made blocking ELISA. Samples obtained at day 9 and at day 11 after infection were scored as positive by approximately half and by all tests, respectively. To score the weak-positive European reference standard (EU2) correctly, 24 h neutralisation tests (positive by 8 of 9 laboratories) and home-made blocking ELISAs (positive by 5 of 6 laboratories) were the most reliable. The results indicate that standardisation is urgently needed to ensure that BHV1-infected animals with low antibody titres are recognised.

Subgrouping of bovine respiratory syncytial virus strains detected in lung tissue

Bovine respiratory syncytial virus is an important respiratory pathogen in cattle. Recently, subgroups of BRSV have been identified, based on antigenic differences. However, little is known about subgroups of BRSV that circulate in the cattle population. Therefore, we determined the reactivity of monoclonal antibodies (mAbs), directed against the G, F, or P protein of BRSV, with lung tissue from 47 calves, that suffered from severe respiratory disease. Fourteen animals (30%) proved to be infected with BRSV, because they all reacted with mAbs against the P or F protein, as detected by fluorescent antibody tests. Monoclonal antibodies against the G protein were able to discriminate between the BRSV-positive specimens: 7 strains were identified as subgroup A strains, and 5 strains as subgroup AB, which is introduced as BRSV subgroup in this paper. Two strains could not be identified unambiguously. It is concluded that BRSV subgroup A and AB were associated with severe respiratory disease, and that strains belonging to either subgroup circulated concurrently in the cattle population in the Netherlands.

Validation of a commercial ELISA for the detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows.

A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested. Compared to the ELISA for sera, the relative specificity and sensitivity of the ELISA for milk samples is 96.5% and 98.9%, respectively when using a S/P% cut-off value of 50% as advised by the manufacturer. The optimal cut-off value was found at S/P% of 90% revealing an optimal specificity (99.0%) combined with an optimal sensitivity (98.1%). Titres in positive individual milk samples ranged from 1 to 2048 with a peak titre of 128. Bulk milk samples contained antibodies with titres ranging from 64 to 512. The ELISA for milk samples was found to be a reliable and robust test. This diagnostic tool is very useful, and may replace the ELISA for serum samples as first choice in order to get insight into the status of lactating individual animals and therewith of the entire herd with respect to BTV infection.

Serological indication for persistence of bovine respiratory syncytial virus in cattle and attempts to detect the virus

SummaryTo identify putative persistent bovine respiratory syncytial virus (BRSV) infections in cattle, seven cattle that had experienced BRSV infections were treated with corticosteroids for two periods of 5 days. During the 5-day periods and the 3 weeks after treatment, attempts were made to isolate BRSV from lung lavage fluid and nasal swab specimens. Fluorescent antibody tests were used to detect BRSV antigen in lung lavage cells. A BRSV specific polymerase chain reaction (PCR) assay was developed, and was performed on lung lavage samples of all seven cattle as well as on various tissues of five of the cattle. In addition, nasal swabs of 74 over-one-year-old cattle, in a closed dairy herd were also assayed by PCR. The virus or its RNA was not detected in putative carriers, by any of the methods used, whereas all positive controls were positive. After corticosteroid treatment, three of the seven cattle showed a four-fold rise in antibody titre, suggesting induction of virus replication. BRSV-seronegative sentinel calves, that were housed together with each corticosteroid-treated animal, did not develop antibodies to BRSV indicating that BRSV was not shed by corticosteroid-treated cattle, or was shed at a very low level. In addition BRSV was not detected in seropositive cattle in a closed farm in summer. Although we consider the rises in antibody titres against BRSV an indication for persistence of BRSV in cattle, BRSV or its RNA was not detected in infected cattle.

Bovine respiratory syncytial virus antibodies in non-bovine species

SummaryTo study the role of non-bovine species in the epidemiology of bovine respiratory syncytial virus (RSV) infections, sera obtained from 9 non-bovine animal species and from humans were examined for bovine RSV specific antibodies. Sera were mainly from animals and humans which had been in contact with cattle. Forty sera of each species were tested in an RSV specific whole virus ELISA as well as in a peptide based ELISA, that was developed to measure antibodies specific for bovine RSV. Antibodies directed against RSV were detected in over 50% of sera obtained from sheep, goat, cattle and human beings, and anti-RSV activity was also found in some roe and dogs and one horse. Antibodies to bovine RSV were found in sera of all tested cattle, 11 (27.5%) goats and in some other individual animals: 3 horses, 2 roe, 1 cat and 1 dog. These results indicate that of the investigated species, besides cattle only goats might play a role in the epidemiology of bovine RSV.