Sara Hägglund

Dynamics of virus infections involved in the bovine respiratory disease complex in Swedish dairy herds

Abstract The dynamics of bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (PIV-3), bovine corona virus (BCoV) and bovine viral diarrhoea virus (BVDV) infections were studied in 118 dairy herds in south western Sweden. By using serology on paired samples from three ∼7 vs. ∼15-month-old calves per herd, the propagation of infections was investigated over about a 1-year period. The results implied that at least 74% of calves had experienced one or more of the monitored infections at the age of ∼7 months (Sample 1, Spring); 30%, 48%, 34% and 8% were seropositive to BRSV, PIV-3, BCoV and BVDV, respectively. Seroconversions to BRSV, PIV-3, BCoV and BVDV occurred in 26%, 38%, 50% and 3% of seronegative animals and 63% had antibodies against two or more infections at ∼15 months (Sample 2). In total, 90–97% of animals that were seropositive in Sample 1 remained positive in Sample 2. A significant association was found between BVDV and BCoV (P =0.01). Moreover, a significantly higher proportion of herds in which no calves had a recorded history of respiratory disease (n =15) were classified as negative to all four infections monitored when compared to herds in which disease was observed (P =0.0002). This study showed a high infection burden in young animals and effective spread of BRSV, PIV-3 and BCoV in one area of Sweden. BVDV infections were restricted to a few herds, reflecting the effect of a voluntary control program against BVDV in Sweden.

Molecular Epidemiology of Bovine Coronavirus on the Basis of Comparative Analyses of the S Gene

ABSTRACT Bovine coronavirus (BCoV), a group 2 member of the genus Coronavirus in the family Coronaviridae, is an important pathogen in cattle worldwide. It causes diarrhea in adult animals (winter dysentery), as well as enteric and respiratory diseases in calves. The annual occurrence of BCoV epidemics in Sweden and Denmark led to this investigation, with the aim to deepen the knowledge of BCoV epidemiology at the molecular level. A total of 43 samples from outbreaks in both countries were used for PCR amplification and direct sequencing of a 624-nucleotide fragment of the BCoV S gene. Sequence comparison and phylogenetic studies were performed. The results showed (i) identical sequences from different animals in the same herds and from paired nasal and fecal samples, suggesting a dominant virus circulating in each herd at a given time; (ii) sequence differences among four outbreaks in different years in the same herd, indicating new introduction of virus; (iii) identical sequences in four different Danish herds in samples obtained within 2 months, implying virus transmission between herds; and (iv) that at least two different virus strains were involved in the outbreaks of BCoV in Denmark during the spring of 2003. This study presents molecular data of BCoV infections that will contribute to an increased understanding of BCoV epidemiology in cattle populations.

A six-year study on respiratory viral infections in a bull testing facility

Abstract Viral infection dynamics and bovine respiratory disease (BRD) treatment rates were studied over six years at a Swedish bull testing station with an ‘all in, all out’ management system. In August of each of the years 1998–2003, between 149 and 185 4–8-month-old calves arrived at the station from 99 to 124 different beef-breeding herds, and remained until March the following year. Only calves that tested free from bovine viral diarrhoea virus (BVDV) were allowed to enter the station and original animal groups were kept isolated from new cattle in their original herds for three weeks before admission. Although neither prophylactic antibiotics, nor BRD vaccines were used, less than 0.7–13.2% (mean 5%) of the calves (n =970) required treatment for BRD during the first five weeks following entry. This was probably due, at least in part, to the season (the summer months) when the animals were commingled. In the six-month period August–February, 38% of the animals were treated one or more times for BRD and mortality was 0.7%. Hereford and Aberdeen Angus calves had significantly higher treatment rates than Charolais, Simmental and Blonde d’Aquitaine. Serological testing on samples obtained in August, November and January indicated that bovine parainfluenza virus 3 (PIV-3) infections occurred each year before November after entry. Bovine coronavirus (BCoV) infections also occurred every year, but in 3/6 years this was not until after November. Bovine respiratory syncytial virus (BRSV) infections occurred only every second year and were associated with a treatment peak and one death on one occasion (December). The herd remained BVDV free during the entire study period. The infection patterns for PIV-3 and BCoV indicated a high level of infectivity amongst bovine calves, whereas the incidence for BRSV was observed at a lower level. Although the rearing of the animals differed from conventional beef production, the study has shown that commingling animals from many sources is not necessarily associated with high morbidity within the first few weeks after arrival. By preventing BRD soon after commingling the prerequisites for protective vaccination at entry might be improved. Applied management routines are discussed.

Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples

Abstract Bovine respiratory syncytial virus (BRSV) causes severe disease in naïve cattle of all ages and is a common pathogen in the respiratory disease complex of calves. Simplified methods for rapid BRSV diagnosis would encourage sampling during outbreaks and would consequently lead to an extended understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format, the turn-around time was shortened drastically and results were obtained with minimal risk for cross-contamination. According to comparative analyses, the detection limit of the fRT-PCR was on the same level as that of a nested PCR and the sensitivity relatively higher than that of a conventional PCR, antigen ELISA (Ag-ELISA) and virus isolation (VI). Interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. Taken together, the data indicated that the fRT-PCR assay can be applied to routine virus detection in clinical specimens and provides a rapid and valuable tool in BRSV research.

Bovine respiratory syncytial virus ISCOMs—Immunity, protection and safety in young conventional calves

Abstract Bovine respiratory syncytial virus (BRSV) is a major cause of bronchiolitis and pneumonia in cattle and causes yearly outbreaks with high morbidity in Europe. Commercial vaccines against this virus needs improvement of efficacy, especially in calves with BRSV-specific maternally derived antibodies (MDA). We previously reported that an experimental BRSV-ISCOM vaccine, but not a commercial vaccine, induced strong clinical and virological protection in calves with MDA, immunized at 7–15 weeks of age. The aim of the present study was to characterize the immune responses, as well as to investigate the efficacy and safety in younger animals, representing the target population for vaccination. Four groups of five 3–8 week old calves with variable levels of BRSV-specific MDA were immunized s.c. twice at a 3 weeks interval with (i) BRSV immunostimulating complexes (BRSV-ISCOMs), (ii) BRSV-protein, (iii) adjuvant, or (iv) PBS. All calves were challenged with virulent BRSV by aerosol 2 weeks later and euthanized on day 6 after infection. The cellular and humoral responses were monitored as well as the clinical signs, the viral excretion and the pathology following challenge. Despite presence of MDA at the time of the immunization, only a minimum of clinical signs were observed in the BRSV-ISCOM group after challenge. In contrast, in all control groups, clinical signs of disease were observed in most of the animals (respiratory rates up to 76min−1 and rectal temperatures up to 41°C). The clinical protection was associated to a highly significant reduction of virus replication in the upper and lower respiratory tract of calves, rapid systemic and local antibody responses and T helper cell responses dominated by IFNγ production. Animals that did not shed virus detectable by PCR or cell culture following challenge possessed particularly high levels of pulmonary IgA. The protective immunological responses to BRSV proteins and the ability to overcome the inhibiting effect of MDA were dependent on ISCOM borne antigen presentation.

Vaccine Safety and Efficacy Evaluation of a Recombinant Bovine Respiratory Syncytial Virus (BRSV) with Deletion of the SH Gene and Subunit Vaccines Based On Recombinant Human RSV Proteins: N-nanorings, P and M2-1, in Calves with Maternal Antibodies

The development of safe and effective vaccines against both bovine and human respiratory syncytial viruses (BRSV, HRSV) to be used in the presence of RSV-specific maternally-derived antibodies (MDA) remains a high priority in human and veterinary medicine. Herein, we present safety and efficacy results from a virulent BRSV challenge of calves with MDA, which were immunized with one of three vaccine candidates that allow serological differentiation of infected from vaccinated animals (DIVA): an SH gene-deleted recombinant BRSV (ΔSHrBRSV), and two subunit (SU) formulations based on HRSV-P, -M2-1, and -N recombinant proteins displaying BRSV-F and -G epitopes, adjuvanted by either oil emulsion (Montanide ISA71VG, SUMont) or immunostimulating complex matrices (AbISCO-300, SUAbis). Whereas all control animals developed severe respiratory disease and shed high levels of virus following BRSV challenge, ΔSHrBRSV-immunized calves demonstrated almost complete clinical and virological protection five weeks after a single intranasal vaccination. Although mucosal vaccination with ΔSHrBRSV failed to induce a detectable immunological response, there was a rapid and strong anamnestic mucosal BRSV-specific IgA, virus neutralizing antibody and local T cell response following challenge with virulent BRSV. Calves immunized twice intramuscularly, three weeks apart with SUMont were also well protected two weeks after boost. The protection was not as pronounced as that in ΔSHrBRSV-immunized animals, but superior to those immunized twice subcutaneously three weeks apart with SUAbis. Antibody responses induced by the subunit vaccines were non-neutralizing and not directed against BRSV F or G proteins. When formulated as SUMont but not as SUAbis, the HRSV N, P and M2-1 proteins induced strong systemic cross-protective cell-mediated immune responses detectable already after priming. ΔSHrBRSV and SUMont are two promising DIVA-compatible vaccines, apparently inducing protection by different immune responses that were influenced by vaccine-composition, immunization route and regimen.

Evaluation of the Immunogenicity of an Experimental Subunit Vaccine That Allows Differentiation between Infected and Vaccinated Animals against Bluetongue Virus Serotype 8 in Cattle

ABSTRACT Bluetongue virus (BTV), the causative agent of bluetongue in ruminants, is an emerging virus in northern Europe. The 2006 outbreak of BTV serotype 8 (BTV-8) in Europe was marked by an unusual teratogenic effect and a high frequency of clinical signs in cattle. Conventional control strategies targeting small ruminants were therefore extended to include cattle. Since cattle were not routinely vaccinated before 2006, the immune responses to BTV have not been studied extensively in this species. With the aims of developing a subunit vaccine against BTV-8 for differentiation between infected and vaccinated animals based on viral protein 7 (VP7) antibody detection and of improving the current understanding of the immunogenicity of BTV proteins in cattle, the immune responses induced by recombinant VP2 (BTV-8) and nonstructural protein 1 (NS1) and NS2 (BTV-2) were studied. Cows were immunized twice (with a 3-week interval) with the experimental vaccine, a commercial inactivated vaccine, or a placebo. The two vaccines induced similar neutralizing antibody responses to BTV-8. Furthermore, the antibody responses detected against VP2, NS1, and NS2 were strongest in the animals immunized with the experimental vaccine, and for the first time, a serotype cross-reactive antibody response to NS2 was shown in cattle vaccinated with the commercial vaccine. The two vaccines evoked measurable T cell responses against NS1, thereby supporting a bovine cross-reactive T cell response. Finally, VP7 seroconversion was observed after vaccination with the commercial vaccine, as in natural infections, but not after vaccination with the experimental vaccine, indicating that the experimental vaccine may allow the differentiation of vaccinated animals from infected animals regardless of BTV serotype. The experimental vaccine will be further evaluated during a virulent challenge in a high-containment facility.

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle

Bluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)-matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n=6) or with adjuvant alone (n=6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes.

Purification, Stability, and Immunogenicity Analyses of Five Bluetongue Virus Proteins for Use in Development of a Subunit Vaccine That Allows Differentiation of Infected from Vaccinated Animals

ABSTRACT Bluetongue virus (BTV) causes bluetongue disease, a vector-borne disease of ruminants. The recent northerly spread of BTV serotype 8 in Europe resulted in outbreaks characterized by clinical signs in cattle, including unusual teratogenic effects. Vaccination has been shown to be crucial for controlling the spread of vector-borne diseases such as BTV. With the aim of developing a novel subunit vaccine targeting BTV-8 that allows differentiation of infected from vaccinated animals, five His-tagged recombinant proteins, VP2 and VP5 of BTV-8 and NS1, NS2, and NS3 of BTV-2, were expressed in baculovirus or Escherichia coli expression systems for further study. Optimized purification protocols were determined for VP2, NS1, NS2, and NS3, which remained stable for detection for at least 560 to 610 days of storage at +4°C or −80°C, and Western blotting using sera from vaccinated or experimentally infected cattle indicated that VP2 and NS2 were recognized by BTV-specific antibodies. To characterize murine immune responses to the four proteins, mice were subcutaneously immunized twice at a 4-week interval with one of three protein combinations plus immunostimulating complex ISCOM-Matrix adjuvant or with ISCOM-Matrix alone (n = 6 per group). Significantly higher serum IgG antibody titers specific for VP2 and NS2 were detected in immunized mice than were detected in controls. VP2, NS1, and NS2 but not NS3 induced specific lymphocyte proliferative responses upon restimulation of spleen cells from immunized mice. The data suggest that these recombinant purified proteins, VP2, NS1, and NS2, could be an important part of a novel vaccine design against BTV-8.

A bovine respiratory syncytial virus model with high clinical expression in calves with specific passive immunity

BackgroundBovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in cattle worldwide. Calves are particularly affected, even with low to moderate levels of BRSV-specific maternally derived antibodies (MDA). Available BRSV vaccines have suboptimal efficacy in calves with MDA, and published infection models in this target group are lacking in clinical expression. Here, we refine and characterize such a model.ResultsIn a first experiment, 2 groups of 3 calves with low levels of MDA were experimentally inoculated by inhalation of aerosolized BRSV, either: the Snook strain, passaged in gnotobiotic calves (BRSV-Snk), or isolate no. 9402022 Denmark, passaged in cell culture (BRSV-Dk). All calves developed clinical signs of respiratory disease and shed high titers of virus, but BRSV-Snk induced more severe disease, which was then reproduced in a second experiment in 5 calves with moderate levels of MDA. These 5 calves shed high titers of virus and developed severe clinical signs of disease and extensive macroscopic lung lesions (mean+/−SD, 48.3+/−12.0% of lung), with a pulmonary influx of inflammatory cells, characterized by interferon gamma secretion and a marked effect on lung function.ConclusionsWe present a BRSV-infection model, with consistently high clinical expression in young calves with low to moderate levels of BRSV-specific MDA, that may prove useful in studies into disease pathogenesis, or evaluations of vaccines and antivirals. Additionally, refined tools to assess the outcome of BRSV infection are described, including passive measurement of lung function and a refined system to score clinical signs of disease. Using this cognate host calf model might also provide answers to elusive questions about human RSV (HRSV), a major cause of morbidity in children worldwide.