Correlations (Spearman’s rho) between relative expression level of selected proteins in bronchoalveolar lavage from calves six days post BRSV infection, BRSV-induced disease and presence of neutrophils in alveolar septa.

<p>Correlations (Spearman’s rho) between relative expression level of selected proteins in bronchoalveolar lavage from calves six days post BRSV infection, BRSV-induced disease and presence of neutrophils in alveolar septa.</p

Scores of neutrophilic infiltration of alveolar septa in BRSV-infected calves.

<p>HE stained sections. a) score 0 (vaccinated calf a, experiment I), b) score 1 (vaccinated calf b, experiment I), c) score 2 (non-vaccinated, BRSV-infected calf l, experiment I) and d) score 3 (non-vaccinated, BRSV infected calf D5, experiment II). Lesions are examples of neutrophil scores and not representative for the overall inflammation regarding exudate and consolidation.</p

Detection of citrullinated histone 3 and myeloperoxidase in serially diluted BAL.

<p>Detection of citrullinated histone 3 (a) and myeloperoxidase (b) by dotblot in bronchoalveolar lavage (BAL) from BRSV-infected calves with clinical signs of disease and high levels of virus shedding (k-o and D1-D5) compared to that in BRSV-ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a-e, Experiment I), or non-vaccinated, non-infected calves (E1-E5, Experiment II). The proteins were selected based on being present in neutrophil extracellular traps, incriminated to be important in the pathogenesis of RSV [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186594#pone.0186594.ref030" target="_blank">30</a>].</p

Animals and experimental design.

<p>Animals and experimental design.</p

Semi-quantification of selected proteins involved in processes that were affected by BRSV-infection.

<p>Proteins were detected in bronchoalveolar lavage from non-vaccinated, BRSV-infected calves with clinical signs of disease and high levels of virus shedding (black bars) and/or in BRSV-ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a, experiment I, grey bars), and/or non-vaccinated, non-infected calves (b, experiment II, grey bars). Proteins were identified by LC-MS/MS and semi-quantified by label-free analysis. Statistically significant differences are indicated by asterisks; p≤0.05(*); p≤0.01 (**); p≤0.001 (***). Proteins were selected based on being related to neutrophil activation and chemotaxis or detoxification of reactive oxygen species: biological processes identified by protein pathway analysis.</p

Proteins associated with BRSV-induced disease.

<p>Proteins associated with BRSV-induced disease.</p

Bronchoalveolar proteomes of calves.

<p>Proteins were identified by LC-MS/MS in bronchoalveolar lavage of calves and were classified as detected at group level if identified in at least 4 out of 5 calves per group. The groups consisted of non-vaccinated, BRSV-infected calves with clinical signs of disease and high levels of virus shedding (a and b, right circles), or ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a, experiment I, left circle), or non-vaccinated, non-infected calves (b, experiment II, left circle). Protein names are provided as supplemental data (*) or in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186594#pone.0186594.t001" target="_blank">Table 1</a> (**).</p

Kinetics of BRSV-neutralising serum antibody titres in cattle of different age and production status.

Cattle were (A) 23–30 months old (6.5 months gestation), (C) 7–11 months old, (D) 4–5 months old, (E) 2–3 months old, not analysed (N.A), or (F) born during or just after a BRSV outbreak in January 2016 (month 0). Limit of detection in the virus neturalising assay: titre 20.</p

Kinetics of HRSV-F-reactive serum antibodies in cattle of different age and production status.

At the time of a BRSV outbreak the cattle were (A) 23–30 months old (6.5 months gestation), (C) 7–11 months old, (D) 4–5 months old, (E) 2–3 months old, or (F) born during or just after the outbreak in January 2016 (month 0). Data are presented as competition percentage. The limit of detection is presented as a dotted line.</p

Kinetics of BRSV-specific serum IgG1 in cattle of different age and production status.

At the time of a BRSV outbreak the cattle were (A) 23–30 months old (6.5 months gestation), (C) 7–11 months old, (D) 4–5 months old, (E) 2–3 months old, or (F) born during or just after the outbreak in January 2016 (month 0). Corrected optical density (COD) values are presented as percentage of a positive control serum.</p