Outcome variables were collected at −48, 0, 4, 8, 24, 32, 48, 72, 96, 120, 144, 168, and 192 h post-treatment (
Figure 1), in addition to the 3-axis accelerometer ear-tags and accelerometers continuously collecting activity and rumination data throughout the study. Outcome variables collected at the given timepoints included rectal temperature, infrared thermography (
IRT) imaging, kinematic gait analysis, mechanical nociception threshold (
MNT), visual analog scale (
VAS) score, clinical illness score (
CIS), computerized stethoscope (
Whisper Veterinary Stethoscope, Merck Animal Health, Madison, NJ) lung score (
CLS), and blood sampling for serum cortisol, substance P, prostaglandin E
2 metabolite (
PGEM), and serum amyloid A (
SAA) analysis. All trained evaluators were blinded to treatment for the duration of the study. Two blinded evaluators scored CIS and VAS, one blinded graduate student operated the computerized stethoscope. Following the 192 h collection, calves were euthanized and transported to the Kansas State Veterinary Diagnostic Laboratory for necropsy and lung lesion scoring.
The IRT images captured the medial canthus of the left eye using a research-grade infrared camera (
Fluke TiX580, Fluke Corp, Everett, WA) as described in Martin et al. (2021) (
link). Infrared images were analyzed using research-specific computer software (
SmartView v. 4.3, Fluke Thermography, Plymouth, MN) to determine maximum and minimum temperatures. The difference between the temperature of the medial canthus baseline and the timepoints following were determined and used for statistical analysis.
A commercially available pressure mat kinematic gait analysis system (Walkway, Tekscan Inc., South Boston, MA) was used to record gait and biomechanical parameters as described in Martin et al. (2021) (
link). The pressure mat was calibrated using a known mass to ensure the accuracy of the measurements at each timepoint. Video synchronization was used to ensure consistent gait between and within calves at each timepoint. Using research-specific software (
Walkway 7.7, Tekscan Inc.), force, contact pressure, impulse, stance time, and stride length were assessed.
A
hand-held pressure algometer (Wagner Instruments, Greenwich, CT) was used for MNT determination. A force was applied perpendicularly, at a rate of approximately 1 kg of force per second, at 1 location on both the left and right side of the ribs of each calf, over the 6th intercostal space for a total of 2 locations, as described in Williams et al., 2020 (
link)). A withdrawal response was indicated by an overt movement away from the applied pressure algometer. Locations were tested three times in sequential order, and the values were averaged for statistical analysis. A second investigator recorded the MNT values to prevent bias by the investigator performing the MNT collection. The collection of MNT values was recorded at only −48, 8, 24, 72, and 192 h timepoints to prevent sensitization of the calf.
A VAS score was assigned by two trained evaluators blinded to treatment allocations using methods adapted from Martin et al. (2020) (
link). The VAS used was a 100 mm (10 cm) line anchored at each end by descriptors of “No Pain” or “Severe Pain.” Seven parameters were used to assess pain: depression, tail swishing or flicking, stance, head carriage, spinal alignment, movement, and ear carriage also adapted from Martin et al. (2020) (
link). No pain was characterized by being alert and quick to show interest, no tail swishing, a normal stance, head carriage above spine level, a straight spine, moving freely around the pen and ears forward. Severe pain was characterized by being dull and showing no interest, more than three tail swishes per minute, legs abducted, head held below spine level, a curved spine, reluctant to move, and ears down. The evaluator marked the line between the two descriptors to indicate the pain intensity. A millimeter scale was used to measure the score from the zero anchor point to the evaluator’s mark. The mean VAS scores of the two evaluators were combined into one score for statistical analysis.
A CIS was assigned by two trained evaluators blinded to treatment allocations. The CIS consisted of: 1) is a normal healthy animal, 2) slightly ill with mild depression or gauntness, 3) moderately ill demonstrating severe depression/labored breathing/nasal or ocular discharge, and 4) severely ill and near death showing minimal response to human approach (Perino and Apley, 1998 (
link)). The CIS scores of the two evaluators were combined into one score for statistical analysis, if either evaluator scored >1 then a final score >1 was assigned, with 1 being considered normal and greater than 1 considered abnormal.
A computerized stethoscope (Whisper, Merck Animal Health, De Soto, KS) was used to analyze lung and heart sounds via a machine-learning algorithm that assigns a CLS from 1 to 5, with 1 being normal and 5 being severely compromised lung tissue (Nickell et al., 2020 (
link)). The bell of the lung stethoscope was placed approximately two inches caudal and dorsal to the right elbow of each calf, and lung sounds were recorded for 8 s. If the recording was deemed acceptable by the computer program, the score was recorded, if not another recording was taken.
Calves were affixed with a 3-axis accelerometer ear-tag (Allflex Livestock Intelligence, Madison, WI) to quantify activity and daily rumination time throughout the study. The average number of minutes spent active or ruminating over 60 min time periods for the study duration was then calculated. An IceTag (IceRobotics Ltd, South Queensferry, Edinburgh, Scotland, UK) accelerometer was also placed on the left rear leg of each calf for the duration of the study. Accelerometer ID was paired with calf ID prior to placement onto the calf. Accelerometers were removed at the conclusion of the study and data were downloaded from the accelerometers for analysis. Steps, standing, lying, lying bouts, and motion index data were collected via the accelerometers and analyzed as described in Martin et al. (2020) (
link).
Rectal temperatures were taken daily by placing a digital thermometer (180 Innovations, Lakewood, CO) against the rectum wall until a temperature reading was produced on the thermometer’s screen.
Blood samples for serum cortisol, substance P, PGEM, and SAA determination were collected from the jugular vein via venipuncture. The whole blood samples were immediately transferred to tubes (Vacutainer, BD Diagnostics, Franklin Lakes, NJ) containing either no additive for cortisol determination or EDTA anticoagulant for PGEM determination. For substance P determination, benzamidine hydrochloride (final concentration of 1mM) was added to EDTA blood tubes 48 h prior to collection. Samples were immediately placed on ice after collection, centrifuged within 30 min of collection for 10 min at 1,500 ×
g, and serum and plasma were placed in cryovials via transfer pipette and stored at −80 °C.
Serum cortisol concentrations were determined using a commercially available radioimmunoassay (
RIA) kit (MP Biomedicals, Irvine, CA) following manufacturer specifications with minor modifications as described in Martin et al. (2021) (
link); the standard curve was extended to include 1 and 3 ng/mL by diluting the 10 and 30 ng/mL manufacturer-supplied standards, 1:10, respectively. The standard curve ranged from 1 to 300 ng/mL. A low (25 ng/mL) and high (150 ng/mL) quality control (
QC) were ran at the beginning and end of each set to determine inter-assay variability. Plain 12 × 75 mm polypropylene tubes were used as blank tubes to calculate non-specific binding. Input for standards, QCs, and samples was adjusted to 50 µL. Samples were incubated at room temperature for 30 min prior to the addition of I-125. Manufacturer instructions were then followed. Tubes were counted on a gamma counter (Wizard2, PerkinElmer, Waltham, MA) for 1 min. The raw data file was then uploaded onto MyAssays Desktop software (version 7.0.211.1238, 21 Hampton Place, Brighton, UK) for concentration determination. Standard curves were plotted as a 4-parameter logistic curve. Samples with a coefficient of variation (
CV) >18% were reanalyzed.
Substance P (SP) concentrations were determined through RIA using methods described by Van Engen et al. (2014) (
link). The standard curve, ranging from 20 to 1,280 pg/mL, was created by diluting synthetic SP (Phoenix Pharmaceuticals, Burlingame, CA) with RIA Buffer (50 mM sodium phosphate dibasic heptahydrate, 13 mM disodium EDTA, 150 mM sodium chloride, 1 mM benzamidine hydrochloride, 0.1% gelatin, 0.02% sodium azide; pH 7.4). A 100 µL of samples, standards, and QCs were aliquoted into plain 12 × 75 mm conical bottom tubes followed by 100 µL of Rabbit anti-SP primary antibody (1:20,000; Phoenix Pharmaceuticals). Iodine-125-SP tracer (custom iodination by PerkinElmer) was diluted with RIA buffer to 20,000 cpm, then 100 µL was added to the samples, standards, and QCs. Samples were then covered and stored at 4 °C for 48 h. At the end of the 48 h incubation, samples were placed on ice and 100 µL of normal rabbit plasma (1:80) and goat anti-rabbit secondary antibody (1:40; Jackson ImmunoResearch, West Grove, PA) were added to each tube. Samples were then incubated at room temperature for 10 min, placed back on ice, and 100 µL of blank bovine plasma was added to the standards and QCs. All tubes then had 1 mL of 12% polypropylene glycol in 0.85% sodium chloride added. Samples were centrifuged at 3,000 ×
g for 30 min at 4 °C and the supernatant aspirated. Tubes were counted on a gamma counter (Wizard2, PerkinElmer, Waltham, MA) for 1 min. The raw data file was then uploaded onto MyAssays Desktop software for concentration determination. Standard curves were plotted as a 4-parameter logistic curve. Samples with a CV >18% were reanalyzed.
PGEM were analyzed using a commercially available ELISA kit (Cayman Chemical, Ann Arbor, MI) following manufacturer specifications with minor modifications as described in Martin et al. (2021) (
link). Sample input was adjusted to 375 µL with 1.5 mL ice-cold acetone added for sample purification. Samples were incubated at −20 °C for 30 min, then centrifuged at 3,000 ×
g for 5 min. The supernatant was transferred to clean 13 × 100 mm glass tubes and evaporated using a CentriVap Concentrator (Labconco, Kansas City, MO) overnight (approx. 18h). Samples were reconstituted with 375 µL of appropriate kit buffer. A 300 µL aliquot of the reconstituted sample was derivatized with proportionally adjusted kit components. Manufacturer protocol was then followed. Samples were diluted at 1:2 and ran in duplicate. Absorbance was measured at 405 nm after 60 min of development (SpectraMax i3, Molecular Devices, San Jose, CA). Sample results were excluded if the raw read exceeded the raw read of the highest standard (Standard 1; 50 pg/mL) or was below the lowest acceptable standard. The lowest acceptable standard was defined for each individual plate and was identified by excluding standards that had a ratio of absorbance of that standard to the maximum binding of any well (%B/B
0) of ≥80% or ≤20%. Any individual sample outside the standard curve, with a %B/B
0 outside the 20%–80% range, or a CV >15% were reanalyzed. PGEM were analyzed at −48, 0, 72, and 192 h timepoints.
SAA concentrations were determined in serum samples using an ELISA assay (Phase Range Multispecies SAA ELISA kit; Tridelta Development Ltd, Maynooth, Kildare, Ireland). Manufacturer specifications were followed and samples were diluted as necessary. Absorbance was measured at 450 nm on a SpectraMax i3 plate reader (Molecular Devices). Raw data were analyzed using MyAssays Desktop software for concentration determination. Standard curves were plotted as a 4-parameter logistic curve. Samples with a CV >15% were reanalyzed.
Flunixin (
FLU; Sigma Aldrich, St. Louis, MO) and flunixin-d3 (
FLU-d3, internal standard, Toronto Research Chemicals, North York, ON, Canada) stock solutions were prepared at 1 mg/mL in methanol and acetonitrile respectively and stored at −80 °C. FLU standard curve and QCs were prepared fresh daily in negative control plasma. The standard curve ranged from 1 to 100 ng/mL. A 50 ng/mL working solution of FLU-d3 was prepared daily by diluting the 1 mg/mL stock in 0.1% formic acid in acetonitrile. Plasma collected in lithium heparin tubes was used for flunixin concentration determination. Samples were extracted via protein precipitation. Briefly, 100 µL of sample, standards, QCs, and blanks were aliquoted and 400 µL of 50 ng/mL FLU-d3 in 0.1% formic acid in acetonitrile was added. Samples were then centrifuged at 3,000 ×
g for 5 min. Supernatant was decanted into 75 × 100 mm glass tubes, evaporated using a CentriVap system (Labconco), and reconstituted with 200 µL of 25% acetonitrile in water. The reconstituted samples were transferred to clean microcentrifuge tubes and centrifuged at 10,000 ×
g for 7 min. An aliquot of 100 µL was transferred to an autosampler vial with a glass insert (QsertVial, Waters Corp., Milford, MA) with pre-slit septum lids. Vials were loaded onto an Acquity H Class ultra-performance liquid chromatography (
UPLC) system coupled with a Xevo TQ-S tandem mass spectrometer (MS/MS; Waters Corp.). Chromatographic separation was achieved using an Aquity UPLC BEH C18 column held at 40 °C during analysis. Mobile phase A and B consisted of 0.1% formic acid in acetonitrile and 0.1% formic acid in 18.2 MΩ.cm water, respectively. Flow rate was set at 0.4 mL/min with the following gradient during the 3 min run time: 30% A at 0–1.49 min, 100% A from 1.5 to 2 min, then 30% A at 2.01 min. The Xevo TQ-S MS/MS was equipped with an electrospray ionization interface set in positive mode. The quantifying transition for FLU was
m/z 297.27→279.24 and the qualifying transition was
m/z 297.27→264.15. The quantifying transition for FLU-d3 was
m/z 300.23→282.26. Data acquisition and analysis were performed using MassLynx and TargetLynx software, respectively (Waters Corp). The standard curve was linear from 1 to 100 ng/mL and the correlation coefficient was accepted if it was at least 0.975. Samples with flunixin concentrations outside the standard curve linear range were diluted at 1:500 with blank plasma and reanalyzed.
All calves were sedated with intravenous xylazine (0.1 mg/kg) and humanely euthanized following the 192 h timepoint with a penetrating captive bolt stunner (CASH Special, FRONTMATEC Accles & Shelvoke Ltd., Minworth, Sutton Coldfield, UK) followed by intravenous injection of potassium chloride (120 mL). A pathologist (K.M.A.), blinded to treatment groups, performed a postmortem examination to determine lung lesions and assign a lung lesion score based on lung consolidation. The lung lesion score was determined using methods described by Fajt et al. (2003) (
link). The equation used was as follows: total percentage lung consolidation = (0.053 × cranial segment of left cranial lobe %) + (0.049 × caudal segment of left cranial lobe %) + (0.319 × left caudal lobe %) + (0.043 × accessory lobe %) + (0.352 × right caudal lobe %) + (0.061 × right middle lobe %) + (0.060 × caudal segment of right cranial lobe %) + (0.063 × cranial segment of right cranial lobe %).
Martin M.S., Kleinhenz M.D., White B.J., Johnson B.T., Montgomery S.R., Curtis A.K., Weeder M.M., Blasi D.A., Almes K.M., Amachawadi R.G., Salih H.M., Miesner M.D., Baysinger A.K., Nickell J.S, & Coetzee J.F. (2021). Assessment of pain associated with bovine respiratory disease and its mitigation with flunixin meglumine in cattle with induced bacterial pneumonia. Journal of Animal Science, 100(2), skab373.
