Bovine rhinitis B virus is highly prevalent in acute bovine respiratory disease and causes upper respiratory tract infection in calves

Abstract

Bovine respiratory disease (BRD) is the most significant cause of cattle morbidity and mortality worldwide. This multifactorial disease has a complex aetiology. Dogma posits a primary viral infection followed by secondary bacterial pneumonia. Bovine rhinitis B virus (BRBV) is an established aetiological agent of BRD, but little is known regarding its pathogenesis. Here, a BRD PCR panel identified 18/153 (11.8 %) lung samples and 20/49 (40.8 %) nasal swabs collected from cattle with respiratory signs as positive for BRBV, which was the most prevalent virus in nasal swabs. Primary bovine tracheal epithelial cells were used to isolate BRBV that was phylogenetically related to contemporary sequences from the USA and Mexico and genetically divergent from the previous sole BRBV isolate. To investigate virus pathogenesis, 1-week-old colostrum-deprived dairy calves were inoculated intranasally with 7.0 log₁₀ TCID₅₀ BRBV. Virus was isolated from nasal swabs, nasal turbinates, trachea and the brain of the challenged animals. Neutralizing antibodies were detected beginning 7 days post-inoculation and peaked at day 14. In situ hybridization (ISH) localized BRBV infection in the upper respiratory ciliated epithelial and goblet cells, occasionally associated with small defects of the superficial cilia lining. Sporadically, pinpoint ISH signals were also detected in cells resembling glial cells in the cerebrum in one calf. Together, these results demonstrate the BRBV infection is highly prevalent in acute BRD samples and while the pathogenicity of BRBV is minimal with infection largely limited to the upper respiratory tract, further research is needed to elucidate a possible initiatory role in BRD.

Keywords: bovine respiratory disease; bovine rhinitis virus; picornavirus.

Fig. 1.

Percentage of nasal swabs and lung tissues submitted for bovine respiratory disease panel PCR that are positive for BRD-associated pathogens. Bovine viral diarrhoea virus, BVDV; bovine herpesvirus 1, BHV1; bovine respiratory syncytial virus, BRSV; Mycoplasma bovis ; bovine coronavirus, BCV; influenza D virus, IDV; Histophilus somni ; Mannheimia haemolytica ; Bibersteinia trehalosi ; Pasteurella multocida ; bovine rhinitis B virus, BRBV.

Fig. 2.

Phylogenetic analysis of the complete genomes of BRBV strains 6900, D2 and B10 and 19 reference genomes. Nucleotide sequences were aligned by clustalw with the phylogenetic tree inferred by maximum-likelihood analysis using the GTR+G+I model of evolution using 1000 bootstrap replicates. BRBV genome sequences determined here are indicated by an asterisk. Scale bar matches that determined in the phylogenetic analysis software.

Fig. 3.

BRBV shedding in nasal swabs detected by (a) qRT-PCR and (b) titration on primary bovine tracheal epithelial cells. Calves 201, 203 and 205 were intranasally inoculated with BRBV on day 0 while calves 204 and 206 were mock inoculated. Calves 201, 203 and 204 were killed on day 5.

Fig. 4.

In situ hybridization staining of BRBV. Positive BRBV staining is visualized in the nasal mucosa (a, b) and trachea (c) of the infected calves (calf 201 and calf 203) and cerebrum (d) in one calf (calf 203). Hybridized signals were detected as pinpoints or granules beside the nuclei (a, b) or diffusely in the cytoplasm (c). Occasionally, small defects of the superficial lining cilia were noted (b). There was an absence of staining in the nasal mucosa (e) and cerebrum (f) of the mock-inoculated calf. Tissues were collected 5 DPI. Cells were counterstained with haematoxylin. The images was taken at 400x (a, b, e), 600x (d, f), and 1000x (c) .