Preparation of bacterial lysate
An isolate of Staphylococcus aureus from a case of bovine mastitis and an isolate of Escherichia coli from a case of bovine diarrhea were kindly provided by the Animal Health Laboratory (AHL), University of Guelph. The E. coli isolate was considered enterotoxigenic based on identification of F41, F5/K99 and STa genes but was not enteropathogenic or verotoxigenic based on absence of eaeA, hlyA, or Shiga toxins 1 or 2 genes). A stock suspension of each isolate was streaked onto a blood agar plate and cultured at 37 °C for 24 h. A single colony of Staphylococcus aureus and of E. coli were each inoculated into separate flasks containing 250 mL tryptose soy broth and incubated at 37 °C with shaking for 16 h (stationary phase). The concentration of viable bacteria (colony forming units (CFU)/mL) was determined from aliquots of the bacterial preparations, by plating serial dilutions onto Columbia agar with 5% sheep blood (Oxoid Canada, Nepean, Ontario, Canada), incubating overnight at 37 °C, and counting the number of bacterial colonies. Other aliquots of the bacterial preparations were killed by incubation in a 65 °C water bath for 90 min. Bacterial killing was confirmed by lack of growth on blood agar. The suspension was centrifuged at 4 °C at 10000 x g for 15 min and washed twice with cold PBS. The bacterial pellets were re-suspended in PBS to create a final stock solution containing 1 × 1011 CFU-equivalents/mL. The heat-killed bacteria were sonicated using a dismembranator (Model 120, Fisher Scientific) at 90% amplitude alternating 20 s pulses with 30 s rest for a total of 5 min. Aliquots of bacterial lysates were stored at − 20 °C. Concentrations of the bacterial lysate are reported as CFU-equivalents.
Cell culture experiments
Primary cultures of bovine tracheal epithelial cells were established as previously described [11, 20]. Once the cells reached 80% confluency, triplicate wells were treated with medium only (negative control) or a combination of 107 CFU-equivalents each of S. aureus and E. coli lysate. Cells treated with 0.1 μg/mL LPS (Sigma Aldrich, MO, USA, L9143) were used as a calibrator. Following treatment, the cells were incubated for 16 h before being harvested for RNA extraction.
RNA extraction and cDNA synthesis were carried out as previously described [20]. Real-time reverse transcription quantitative PCR was used to evaluate the effect of bacterial lysate on TAP and LAP expression relative to expression of the reference gene GAPDH as previously described [10, 11, 20]. Stability of expression of GAPDH across treatments was assessed using ANOVA. Template-negative wells were included in each run, and data of technical triplicate samples were averaged. The specificity of the reaction was confirmed by examining melting curves and identification of a single peak at approximately 82.8 °C (TAP), 86.9 °C (LAP) and 85.4 °C (GAPDH).
Aerosolization of bacterial lysate to calves
Use of animals in these studies was approved by the Animal Care Committee of the University of Guelph (AUP #3286) according to Canadian Council on Animal Care guidelines. Calves were obtained from the Elora Dairy Research Centre (Ontario Ministry of Agriculture, Food and Rural Affairs). Eight 3-month-old Holstein bull calves were housed in pairs within climate-controlled, biosecurity level 2 pens in an isolation facility. Shavings were used as bedding, and hay, water and calf starter pellets (Sharpe Farms Supplies Limited, Guelph, Ontario) were offered ad libidum.
An aerosol of S. aureus and E. coli lysate was administered to 6 calves. Calves were randomly assigned to receive either 108 (calf 1), 109 (calf 2), 1010 (calf 3), 1011 (calf 4) or 1012 (calves 5 and 6) CFU-equivalents suspended in a total volume of 10 mL PBS. The bacterial lysates or PBS were delivered by aerosol using a compressor (Precision Medical, Northampton, PA) that produced approximately 25 psi of air pressure and was attached to a Whisper Jet nebulizer (Marquest, Englewood, Colo) connected to a small Equine Aeromask (Trudell Medical International, London, ON) placed over the calf’s muzzle. A prior study showed that this system delivered aerosol to the upper respiratory tract as well as the bronchioles and alveoli (Bassel et al., 2019). Personnel wore powered air-purifying respirators (PAPRs) and N95 respirators were placed over the one-way exhalation valves on the calf masks to reduce room air contamination. Two control calves (calves C1 and C2) were randomly assigned to receive only PBS, to ensure that the aerosolization procedure did not affect the measured parameters.
Clinical and hematologic responses
Calves were assessed prior to and at 1, 2, 4, 6, 12, 18 and 24 h following administration of aerosolized bacterial lysate or PBS without knowledge of treatment group. At these times, rectal temperature, respiratory rate and effort, heart rate, and mentation were assessed. Additional clinical signs including lack of appetite, coughing or nasal discharge were noted when present. Clinical scores were assigned as described in Additional file 11. Blood was collected into sodium citrate anticoagulant for measuring plasma fibrinogen and without anticoagulant for evaluation of serum haptoglobin (AHL, University of Guelph) prior to and 24 h following aerosolization of bacterial lysate or PBS.
Bronchoalveolar lavage fluid analysis
Bronchoalveolar lavage fluid was collected from all calves prior to and 24 h following administration of the bacterial lysate. Calves were sedated with xylazine and BALF was collected from the right caudodorsal lung for the baseline sample collection and the left caudodorsal lung for the post-aerosol sample collection, by infusing 120 mL sterile saline (120 mL, 0.9%) through an endoscope and retrieving the lavage fluid by manual suction through a 60 mL syringe. The sample was placed on ice until further processing (within 2 h). Samples were filtered through gauze prior to determination of total nucleated cell counts using electrical impedance (Z2 Coulter counter, Beckman Coulter, Mississauga, ON) and cytocentrifuge preparations were prepared using 200 μL of fluid. Differential cell counts were performed on 200 cells on the Wright-stained slide preparations. Olympus CellSens software was used to trace the outline of individual cells and measure the area of 15 macrophages in a single central 400x field.
Mass spectrometry analysis
For mass spectrometry, the remaining BALF was centrifuged (500×g, 5 min, 4 °C), then desalted and concentrated by centrifugal filtration (Amicon® Ultra-15, Millipore, Bedford, MA, USA). Protein concentrations were measured using a Bradford assay [35]. Tandem mass tag (TMT) mass spectrometry (SPARC BioCentre, The Hospital for Sick Children, Toronto, Canada) was conducted on four pre- and post-aerosolization samples (calves 1–4). The normalized protein concentrations were reported. Samples were reduced, alkylated, digested, and TMT labelled according to manufacturer’s directions (Thermo Fisher TMT 10 Plex, Product 90,110). Labelled peptides from all samples were combined and lyophilized. Peptides were cleaned up using a C18 ZipTip (Millipore) and then lyophilized.
Samples were analyzed on a Thermo Scientific Orbitrap Fusion-Lumos Tribid Mass Spectrometer (ThermoFisher, San Jose, CA) outfitted with a nanospray source and EASY-nLC 1200 nano-LC system (ThermoFisher, San Jose, CA) and equipped with ETD mode as described in Additional file 12. Data analysis was performed using Proteome Discoverer 2.2 against a Uniprot Bovine Database (6002 sequences). TMT modifications of lysine and the peptide N-termini as well as carbamidomethyl of cysteine were considered fixed modifications while oxidation of methionine and protein N-terminal acetylation were considered variable. Parent mass tolerance was set to 10 ppm, fragment mass tolerance was set to 0.6 Da. Reporter ion quantification for the 8 TMT channels was done on the MS3, and lot-specific correction factors were used. Proteins that were significantly increased or decreased following lysate treatment were analysed in PANTHER v14.1 (www.pantherdb.org/) to analyze function. The pathways overrepresented among the upregulated and down regulated proteins were analyzed (Reactome version 68, www.Reactome.org; [37]) using a Fisher’s exact test to produce a probability score that was corrected for false discovery rate using the Benjamani-Hochberg method.
Cytokine analysis
The following cytokines were measured in BALF: IFN-α, IL-8, IL-2, IL-6, tumour necrosis factor alpha (TNF-α), IFN-γ, IL-10, and IL-17 using a multiplexed electrochemiluminescent ligand-binding assay with the U-plex assay platform (MSD, Rockville, MD). The assay was developed using two separate Meso Scale Diagnostics (MSD) U-Plex panels, following the MSD U-Plex protocol guide. Cytokines were quantified using the U-plex assay platform (MSD, Rockville, MD) assembled according to manufacturer’s instructions using a chemiluminescent readout. Panel 1 included biotinylated capture antibodies: IL-1β (Biorad, Hercules, CA), IL-2 (R&D Systems Minneapolis, MN), IL-6 (R&D Systems), IFN-α (Kingfisher Biotech, Minneapolis, MN), and TNF-α (R&D Systems). Panel 2 included: IL-8 (Mabtech, Cincinnati, OH), IL-10 (Biorad), IL-17A (Mabtech), and IFN-γ (Mabtech). The IL-1β antibody was biotinylated using EZ-Link Sulfo-NHS-Biotin (Thermo Scientific) at a challenge ratio of 1:20 according to manufacturer instructions.
All calibrators used in the assay were purchased from Kingfisher Biotech. The panel 1 calibrators were diluted to a concentration of 40,000 pg/mL in 1X Dulbecco’s PBS without calcium or magnesium, followed by 4-fold serial dilutions into 1X DPBS. The panel 2 calibrators were diluted to a concentration of 10,000 pg/mL in 1X DPBS followed by 4-fold serial dilutions.
The detection antibodies were sulfo-tagged following the MSD quick guide conjugation protocol using a challenge ratio of 1:20. Panel 1 detection antibodies were used at the following concentrations: IL-1β at 1 μg/mL (Biorad), IL-2 at 0.5 μg/mL (R&D Systems), IL-6 at 2 μg/mL (R&D Systems), IFN-α at 1 μg/mL (Kingfisher Biotech) and TNF-α at 0.5 μg/mL (R&D systems). Panel 2 detection antibodies were used at the following concentrations: IL-8 at 0.5 μg/mL (Mabtech), IL-10 at 1 μg/mL (Biorad), IL-17A at 0.2 μg/mL (Kingfisher Biotech) and IFN-γ at 1 μg/mL (Mabtech).
Cytokines were quantified using the U-plex assay platform (MSD, Rockville, MD) assembled according to manufacturer’s instructions using a chemiluminescent readout (Additional file 12). For soluble protein levels, a BCA protein kit (Thermofisher, Rockford, IL) was used. The cytokine concentrations were normalized to the total protein levels for each sample.
Post-mortem examination
All but one of the lysate-treated calves were euthanized at 24 h following administration of aerosols, by intravenous injection of pentobarbital. Gross postmortem examination was performed within 2 h of death including visual inspection and palpation of respiratory tissues. The lungs (trachea removed) and heart were weighed and the lung: heart weight ratio was calculated for each calf. Samples of nasal mucosa, trachea and cranioventral, caudodorsal and caudoventral regions of lung were placed in 10% formalin, and histologic sections were routinely prepared and stained with hematoxylin and eosin.
Statistical analysis
Data were compared between samples obtained before and after aerosolization of bacterial lysate within the same animal. Descriptive statistics, t-tests and ANOVA were performed (Graphpad Prism v8.0, San Diego, CA, USA) and 2-sided tests were considered significant when P < 0.05. Outcome variables were evaluated for normality using a D’Agostino-Pearson omnibus K2 test, Shapiro-Wilk test and Kolmogorov-Smirnov test and transformed as indicated. Grubb’s test, with alpha set at 1%, was used to detect outliers, which were subsequently removed from further analysis.
ANOVA compared the effects of bacterial lysate on normalized ratios of TAP: GAPDH and LAP: GAPDH expression in cultured tracheal epithelial cells. Repeated measures of clinical parameters including rectal temperatures, respiratory rate, and heart rate were evaluated using repeated measures one-way ANOVA with a Geisser-Greenhouse correction to adjust for unequal variability of differences. Multiple comparisons of post-aerosolization values against baseline (time 0) values were evaluated using a Dunnett’s test. Residuals were evaluated to determine whether ANOVA assumptions were met. For clinical scores, a repeated measure non-parametric Friedman test was conducted, with post hoc Dunn’s tests to compare post-aerosolization clinical scores with baseline.
Paired t-tests were used to compare baseline and post-stimulation concentrations of serum haptoglobin, plasma fibrinogen, hematology values and BALF parameters (cell counts and cytokines). For the cytokine assays, analytes that were below the limit of detection were assigned a value that was equal to the lower limit of detection divided by the square root of 2 [36]. Outcome variables that were not normally distributed and were not normalized following transformation were assessed non-parametrically using a Wilcoxon matched-pairs sign rank test.
Two-sided tests with α < 0.05 were considered significant. Data from the study is provided as Additional file 13.