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

Abstract

Background: Bovine 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.

Results: In 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.

Conclusions: We 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.

Figure 1

Daily and accumulated clinical score following aerosol challenge with either BRSV-Snk or BRSV-Dk. Six calves were experimentally infected with virulent BRSV, either passaged in vivo (BRSV-Snk, n = 3, calves A1-3), or in vitro (BRSV-Dk, n = 3, calves B1-3). Following infection on post-infection day (PID) 0, calves were monitored for seven days. Daily clinical scores (panel A) were calculated from observed clinical signs (see Tables 1 and 2). Accumulated clinical scores, from PID 0 to PID 7 (panel B), were calculated as the area under individual clinical score curves.

Figure 2

Pulmonary pathology and neutrophil influx in calves following aerosol challenge with either BRSV-Snk or BRSV-Dk. Calves were experimentally infected as described in Figure 1. Calves were euthanized seven days after infection and the macroscopic extent of lung lesions were documented (panel A). From each calf, trachea tissue and four lung tissue samples were collected for sectioning, staining and histopathological description and scoring of severity of inflammation (1–3). Panels B:I-IV show representative HE-stained sections from: (B:I) trachea from calf A2; (B:II) trachea from calf B3; (B:III) lung from calf A2; and (B:IV) lung from calf B3. Horizontal bars indicate 50 μm in panels B:I and B:II, and 100 μm in panels B:III and B:IV. Mean histopathological severity of inflammation per calf, is shown on the x-axis in panel C, along with the proportion (%) of macroscopic lung lesions per calf on the y-axis. Bronchoalveolar lavage (BAL) was performed on PID −1 and PID 7, and cell types in BAL samples enumerated (Panel D). Stacks represent the mean total number of cells in BAL per ml, with associated standard deviation, as well as the number of neutrophils, macrophages and lymphocytes in BAL per ml. The proportion of eosinophils were <1% in all samples.

Figure 3

Virus detected in the airways of calves after aerosol challenge with either BRSV-Snk or BRSV-Dk. Calves were experimentally infected as described in Figure 1. Daily nasal secretion (NS) samples were collected for eight consecutive days, starting on PID 0. After euthanization on PID 7, bronchoalveolar lavage (BAL) was collected, along with tissue samples from the trachea and lung, for histopathology and immunohistochemistry (IHC) to demonstrate BRSV-antigen (brown stain). BRSV RNA in daily NS (panel A) and BAL (x-axis, panel B) was detected by RT-qPCR, and is expressed as log₁₀ TCID₅₀ equivalent unit. The accumulated virus shed in NS (y-axis, panel B) was calculated as the area under individual curves. Panels C:I-IV show representative IHC-stained sections of: (C:I) trachea from calf A2; (C:II) trachea from calf B3; (C:III) lung from calf A2; and (C:IV) lung from calf B2. Horizontal bars in panels C:I-IV indicate 50 μm.

Figure 4

Serum anti-BRSV IgG ₁ in calves, before and after aerosol challenge with either BRSV-Snk or BRSV-Dk. Calves were experimentally infected as described in Figure 1. BRSV-specific IgG₁ antibodies, detected by ELISA (SVANOVIR® BRSV-Ab ELISA, Boehringer Ingelheim Svanova, Sweden) in serum diluted 1:25, are expressed as percent of the corrected optical density (COD) of a positive control sample. The shaded area of the chart indicates ≤10% COD of positive, defined as negative by the kit manufacturer.

Figure 5

Clinical, pathological and virological ranking of calves following aerosol challenge with either BRSV-Snk or BRSV-Dk. Calves were experimentally infected as described in Figure 1. Following challenge, calves were ranked (panel A) based on accumulated daily clinical scores (Clinical rank), nasal virus shed (Viral-shed rank), and extent of lung lesions (Pathology rank). Panel B shows the rank sum for each of the three ranks, and the total rank sum per group.

Figure 6

BRSV-Snk challenge in calves with low or moderate passive immunity: clinical, virological and pathological outcomes. Experimental challenge by inhalation of aerosolized BRSV passaged in gnotobiotic calves (BRSV-Snk) was performed in three calves (A1-3; study 1) with low levels of BRSV-specific maternal antibodies (MDA), and later reproduced in five calves (C1-5; study 2) with moderate levels of MDA. Clinical scores (Panel A) and BRSV RNA detected by RT-qPCR in nasal swab samples (Panel B), from the day of challenge (post-infection day or PID) 0 to PID 7. Individual values are presented for calves C1-5, and mean values are presented for calves A1-3. For both groups of calves, panel C shows mean extent of macroscopic lung lesions on the y-axis, expressed as a percent of total lung area, and the mean severity of histopathological inflammation, scored from 0 to 3, on the x-axis. For mean values, vertical and horizontal lines indicate standard deviation.

Figure 7

BRSV-Snk challenge in calves with low or moderate passive immunity: effect on airway resistance and reactance. Experimental challenge by inhalation of aerosolized BRSV passaged in gnotobiotic calves (BRSV-Snk) was performed in five calves with moderate levels of BRSV-specific maternal antibodies (MDA). Lung function was measured using the forced oscillation technique, and a tightly fitting face mask; before challenge on post-infection day (PID) 0, and after challenge, on PID 6. Resistance (Panel A) and reactance (Panel B) at 10Hz were calculated and presented as kPa/L/s.

Figure 8

Variations of cells populations and cytokines in bronchoalveolar lavage (BAL) from calves challenged with BRSV-Snk. Experimental challenge by inhalation of aerosolized BRSV passaged in gnotobiotic calves (BRSV-Snk) was performed in five calves with moderate levels of BRSV-specific maternal antibodies (MDA). Samples of the cells populating the lower airways were collected via BAL seven days after infection, and in addition, from three healthy uninfected calves (Control). Cells in BAL samples were analyzed by light microscopy (panel A), and expressed as group mean ×10⁶ cells/ml. The concentrations of indicated inflammatory cytokines in BAL sample supernatants were measured using specific ELISAs, and are expressed in ng/ml: (panel B) interleukin (IL)-4; (panel C) IL-6; (panel D) IL-8; (panel E) interferon gamma (IFNγ); and (panel F) tumor necrosis factor alpha (TNFα). Individual data are indicated by rings and group means by horizontal lines. Probability (p) of statistically significant differences between groups is given in each panel, where p ≤ 0.05 was considered statistically significant.