Bovine respiratory disease is the leading cause of morbidity and death in US beef feedlot cattle, and approximately 55.9% of US beef feedlots report administering cattle with BRD an NSAID in conjunction with an antimicrobial.1 Historically, flunixin meglumine was the only NSAID available in an injectable formulation that was approved by the FDA for use in cattle. The injectable formulation of flunixin meglumine is approved for IV administration to control pyrexia and inflammation associated with BRD and endotoxemia and has a labeled slaughter withdrawal time of 4 days. Extravascular administration of flunixin meglumine causes severe localized tissue damage,2 and flunixin meglumine was the second leading violative residue identified in US meat in 2016.3 In July 2017, the FDA approved a novel TDFM formulation that can be administered to cattle as a pour-on solution to control pyrexia associated with BRD and alleviate pain associated with interdigital phlegmon (foot rot).4 Prior to the approval of TDFM, administration of flunixin meglumine was only approved for cattle via the IV route, making it difficult for cattle producers to administer. The TDFM formulation allows cattle producers an easier way to administer flunixin meglumine to provide pain and fever relief for cattle, which could improve compliance with on-label administration and decrease the number of violative flunixin meglumine residues in beef.
In cattle with experimentally induced inflammatory exudate tissue, topical administration of TDFM decreases synthesis of PGE2,5 which suggests that, as expected of an NSAID, TDFM inhibits production of prostaglandins through the arachidonic acid pathway. The TDFM is effectively absorbed through the skin of cattle6 and alleviates BRD-induced pyrexia for 6 hours after administration.4 The purpose of administering an NSAID to cattle with BRD is to alleviate pyrexia and reduce the harmful effects of disease-induced inflammation, particularly in the respiratory tract.7 Although NSAIDs are consistently effective in alleviation of pyrexia, their effects on inflammatory biomarkers have been infrequently studied, and the results of those studies vary.8–10 The objective of the study reported here was to quantify the acute immunologic and metabolic responses of beef heifers following topical administration of TDFM at various times relative to a respiratory pathogen challenge.
Materials and Methods
Animals
All experimental procedures were approved by the Institutional Animal Care and Use Committee of the USDA Agricultural Research Service Livestock Issues Research Unit (LIRU IACUC No. 1605F) and were conducted in compliance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching.11 Thirty-two British crossbred beef heifers with a mean ± SD body weight of 170 ± 21.1 kg (range, 127 to 213 kg) were obtained from a commercial feedlot in southwest Kansas and transported approximately 529 km to the USDA Agricultural Research Service Livestock Issues Research Unit's Research Complex in New Deal, Tex. Study heifers were selected from a larger group of heifers on the basis of uniformity in body weight and had not received any antimicrobials during the 60 days prior to selection. Each heifer was administered an intranasal vaccinea against BHV1, parainfluenza virus type 3, and bovine respiratory syncytial virus at birth and a multivalent modified-live virus vaccineb against BHV1, bovine viral diarrhea virus types 1 and 2, bovine respiratory syncytial virus, and parainfluenza virus type 3, SC, at approximately 60 days old. No other vaccines were administered prior to study initiation (approx 140 days). Heifers had ad libitum access to water and a customized complete feedc with a 14% crude protein content throughout the study.
Study design
All heifers were experimentally inoculated with BHV1 and Mannheimia haemolytica. Experimental inoculation (challenge) with M haemolytica was designated as time 0.
Cattle were assigned to 1 of 4 treatment groups such that there were 8 heifers/group. Treatment group assignment was based on the order in which the heifers passed through the chute after they were allowed to commingle in an alley. Heifers in the control group did not receive TDFM. For heifers in the other 3 groups (groups A, B, and V), 1 dose of TDFMd (3.3 mg/kg) was administered at various times relative to BHV1 and M haemolytica challenges. Heifers in group A received TDFM the day after arrival at the research center (−144 hours). Heifers in group V received TDFM at the time of BHV1 challenge inoculation (−72 hours), and heifers in group B received TDFM at the time of M haemolytica challenge inoculation (0 hours). The drug was topically applied with a single-use polystyrene syringe along the dorsal mid-line starting at the shoulders and ending at the tail head in accordance with the label directions.
Study protocol
At research center arrival, the heifers were housed as a single group in a dirt pen (7.6 × 18.3 m) with access to shade and water and allowed to rest overnight. The next morning (−144 hours), the heifers were allocated to treatment groups and processed through a chute system where they were individually weighed and restrained for blood sample collection. All heifers were instrumented with a vaginal temperature-recording device as described,12 which recorded the temperature every 5 minutes until it was removed 72 hours after M haemolytica challenge.
Heifers assigned to group A were also administered TDFM. The heifers were then returned to 4 outdoor dirt pens where they were group housed (8 heifers/group) in accordance with treatment group assignment.
Each heifer was assigned clinical illness scores for cough, nasal discharge, ocular discharge, and posture (Appendix) after it was returned to its assigned pen and daily thereafter at approximately 8 am. The scoring system was adapted from a system13 developed for dairy calves, and all scores were assigned by 1 of 2 trained personnel throughout the study. Any heifer deemed to be suffering was immediately removed and treated. One animal was removed from the trial at hour 48 because of severe clinical illness characterized by tachypnea and profuse salivary discharge from the mouth. The attending veterinarian was contacted and recommended tildipirosin (4 mg/kg, SC, once) administration immediately. That heifer was removed from the study at that time and housed in the outdoor pen with access to food and water until recovery.
At −72 hours, all heifers were processed through the chute system again, where they were individually weighed and restrained for blood sample collection, assignment of a nasal lesion score, and challenge inoculation with BHV1. Heifers in group V were also administered TDFM. Nasal lesions were scored on a scale of 0 to 4, where 0 = no visible lesions within the nares, 1 = presence of lesions on < 10% of the visible mucosa within the nares, 2 = presence of lesions on 11% to 25% of the visible mucosa within the nares, 3 = presence of lesions on 26% to 50% of the visible mucosa within the nares, and 4 = presence of lesions on > 50% of the visible mucosa within the nares. The BHV1 challenge strain was a standard (Colorado) strain of the virus obtained from the National Veterinary Services Laboratory in Ames, Iowa. For each heifer, 1 vial of BHV1 was diluted with 1.4 mL of sterile saline (0.9% NaCl) solution to yield 2 mL of inoculum with a BHV1 concentration of approximately 1 × 108 PFUs/mL. Then 1 mL of the inoculum was administered into each nares by use of a syringe and nasal atomization device.e
At 0 hours, all heifers were processed through the chute system, where they were individually weighed and restrained for assignment of a nasal lesion score as previously described, aseptic placement of an indwelling jugular catheter, and challenge inoculation with M haemolytica. Heifers in group B were also administered TDFM. An M haemolytica serotype A1 isolate obtained from a laboratory was used as the challenge strain. A mean dose of 1.18 × 106 CFUs of logarithmic-phase M haemolytica was diluted with 50 mL of sterile PBS solution. The resulting inoculum was administered intratracheally. Briefly, the M haemolytica inoculum was injected into the trachea approximately 15 cm distal to the pharynx. The head was immobilized with a halter, and the skin overlying the intended injection site and adjacent area was clipped to remove the hair and aseptically prepared with dilute betadine solution and 70% ethanol. A 16-gauge needle was used to inject the entire volume of inoculum into the trachea.
Following challenge inoculation with M haemolytica, each heifer was individually housed in a stall (length, 2.28 m; width, 0.76 m; and height, 1.67 m) for 72 hours to facilitate blood sample collection. Blood samples were collected at 0 (immediately after), 2, 4, 6, 8, 12, 24, 36, 48, 60, and 72 hours after M haemolytica challenge. For each heifer following collection of the blood samples 72 hours after M haemolytica challenge, body weight was recorded, the jugular catheter and vaginal temperature-recording device were removed, and tildipirosinf (4 mg/kg, SC, once) was administered in accordance with the product label. Individual-heifer water consumption was monitored by use of a watering bowlg while heifers were housed in stalls. The heifers were then returned to their assigned pens. At 144 and 360 hours, they were processed through the chute system and restrained for blood sample collection.
Blood sample collection and processing
All blood samples were obtained by jugular venipuncture except for those collected between 0 and 72 hours after M haemolytica challenge while the heifers were individually housed in stalls, which were obtained via the indwelling jugular catheter. Blood (approx 4 mL) for a CBC and determination of phagocytic and oxidative burst capacities was collected into a blood collection tubeh that contained 1.7 mg of potassium EDTA as an anticoagulant (EDTA tube) at −144, −72, 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, and 72 hours after M haemolytica challenge. Blood (approx 4 mL) for determination of L-selectin expression on the surface of neutrophils was collected into a 4-mL blood collection tubeh that contained 75 U of sodium heparin as an anticoagulant (heparin tube) at −144, −72, 0, and 72 hours after M haemolytica challenge. Blood (approx 9 mL) for the acquisition of serum to determine cortisol, glucose, NEFA, SUN, IL-1β, IL-4, IL-6, IFNγ, and haptoglobin concentrations and anti-BHV1 antibody titers was collected into a 9-mL blood collection tubei with no additives (plain tube) at −144, −72, 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, 72, 144, and 360 hours after M haemolytica challenge. Blood samples collected into plain tubes were allowed to clot at room temperature (approx 22°C) for 30 minutes after collection and then were centrifuged at 1,500 × g and 4°C for 20 minutes. Serum was harvested from each sample, placed in individual cryotubes, and stored frozen at −80°C until analyzed.
For blood samples collected into EDTA tubes at −144, −72, 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, and 72 hours after M haemolytica challenge, CBCs were performed by use of an automated hematology analyzerj and bovine-specific algorithms.
Blood samples collected into EDTA tubes at −144, −72, 0, and 72 hours after M haemolytica challenge were also evaluated to determine the phagocytic and oxidative burst capacities of neutrophils against Escherichia coli. Briefly, for each sample, 200 μL of EDTA-anticoagulated blood was chilled in an ice bath, and then 40 μL of 100μM dihydrorhodamine and 40 μL of a solution containing heat-killed E coli (concentration, 1 × 106 CFUs/mL) were added to the blood sample. The resulting solution was incubated in a warm water bath (38.5°C) for 10 minutes, then placed in an ice bath for 15 minutes to stop the reaction, hypotonically lysed, and washed with ice-cold PBS solution. Each batch of samples run included a negative control, which consisted of the E coli solution only. The negative control samples remained on ice throughout the 10 minutes that test samples were incubated with dihydrorhodamine but were otherwise processed in the same manner as the test samples. For each sample, a flow cytometerk was used to analyze leukocytes, and neutrophils were gated on a scatterplot of forward scatter × side scatter. Results were reported as the geometric mean fluorescence intensity; the greater the intensity, the greater the extent of phagocytosis and oxidative burst (ie, neutrophil functionality).
Blood samples collected in heparin tubes at −144, −72, 0, and 72 hours after M haemolytica challenge were analyzed for expression of the adhesion molecule L-selectin (CD62L) on the surface of neutrophils as described.14 Briefly, for each heparin-anticoagulated blood sample, 50 μL of blood was incubated with sufficient anti-bovine CD62L antibodyl to achieve an end dilution of 5 μg/mL for 1 hour in an ice bath. A negative control sample that consisted of only the anti-bovine CD62L antibody was processed in the same manner as the test samples. Following incubation, each sample was washed twice with PBS solution and analyzed with flow cytometry as described for determination of phagocytic and oxidative burst capacities of neutrophils. The mean fluorescence intensity for each sample was recorded.
All serum analyses were performed in duplicate for each sample, and mean values were calculated and used for analysis purposes. Serum samples obtained from blood samples collected at −144, −72, 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, 72, and 144 hours after M haemolytica challenge were analyzed for determination of cortisol, glucose, NEFA, SUN, IL-1β, IL-4, IL-6, and IFNγ concentrations. Serum samples obtained from blood samples collected at −144, −72, 0, 4, 8, 12, 24, 36, 48, 60, 72, 144, and 360 hours after M haemolytica challenge were also evaluated for haptoglobin concentration. Serum samples obtained from blood samples collected at −144 and 360 hours after M haemolytica challenge were submitted to the Texas Veterinary Medical Diagnostic Laboratory in Amarillo, Tex, for determination of the anti-BHV1 antibody titer by means of a virus neutralization assay.
Serum cortisol concentration was determined by use of a commercially available enzyme immunoassay kitm in accordance with the manufacturer's instructions. The cortisol concentration in test samples was compared with standard curves that were generated for standards with known cortisol concentrations. The intra-assay and interassay coefficients of variation for the serum cortisol assay were 6.9% and 18.2%, respectively.
Serum haptoglobin concentration was determined by use of a commercially available enzyme immunoassay kitn in accordance with the manufacturer's instructions. The intra-assay and interassay coefficients of variation for the serum haptoglobin assay were 10.5% and 31.8%, respectively.
Serum glucose concentration was determined by use of a commercially available enzymatic methodo that was modified to fit a 96-well format. Briefly, 2 μL of serum or a prepared standard was added to each well of a 96-well plate, and then 300 μL of prepared working solution was added to each well. The plate was incubated at 37°C for 5 minutes and then read with a plate reader at a frequency of 505 nm. The glucose concentration in test samples was compared with standard curves that were generated for standards with known glucose concentrations. The intra-assay and interassay coefficients of variation for the serum glucose assay were 13.3% and 18.0%, respectively.
Serum NEFA concentration was determined by use of a commercially available enzymatic colorimetric assayp that was modified to fit a 96-well format. Briefly, 5 μL of serum or a prepared standard was added to each well of a 96-well plate, and then 200 μL of a color reagent (A) was added to each well. The plate was incubated at 37°C for 5 minutes and read with a spectrophotometer at a frequency of 550 nm. Then 100 μL of another color reagent (B) was added to each well of the plate. The plate was incubated at 37°C for another 5 minutes and read with a plate reader at a frequency of 550 nm. For each sample, NEFA concentration was determined by multiplying the absorbance value for the first reading by 0.67 to account for the change in volume and subtracting that product from the absorbance value for the second reading. The NEFA concentration in test samples was compared with standard curves that were generated for standards with known NEFA concentrations. The intra-assay and interassay coefficients of variation for the serum NEFA assay were 8.5% and 14.0%, respectively.
The SUN concentration was determined by use of a colorimetric assaym in accordance with the manufacturer's instructions. The SUN concentration in test samples was compared with standard curves that were generated for standards with known SUN concentrations. The intra-assay and interassay coefficients of variation for the SUN assay were 5.8% and 23.9%, respectively.
Serum concentrations of IFNγ, IL-4, and IL-6 were determined by use of a custom-purchased bovine-specific 3-plex sandwich-based ELISA kit,q and the serum concentration of IL-1β was determined by use of a commercially available bovine-specific ELISA kit.q For all cytokine assays used, the intra-assay coefficients of variations were < 11.2%, and the interassay coefficients of variation were < 13.6%.
Data analysis
For each heifer, vaginal temperature was recorded every 5 minutes from −72 to 72 hours after M haemolytica challenge, and the mean vaginal temperature was calculated for each 1-hour interval for analysis purposes. Also for each heifer, the change in vaginal temperature after M haemolytica challenge was compared with the baseline vaginal temperature (mean vaginal temperature from −144 to −72 hours [prior to any immunologic challenge]).
The data distribution for each continuous outcome of interest (hematologic variables, serum biochemical and cytokine concentrations, vaginal temperature, and water intake between 0 and 72 hours after M haemolytica challenge) was assessed for normality by means of the Shapiro-Wilk test. Mixed linear models were used to assess the effect of administration of TDFM and time relative to M haemolytica challenge on hematologic variables, serum biochemical and cytokine concentrations, vaginal temperature, and water intake between 0 and 72 hours after M haemolytica challenge. Each model included fixed effects for treatment group (treatment), sample acquisition time (time), and the interaction between treatment and time and a random effect to account for repeated measures within each heifer. For each outcome, models with various covariance structures were compared by means of the Akaike information criterion, and results for the model with the covariance structure that yielded the lowest Akaike information criterion value were reported. Either the compound symmetry or autoregressive 1 covariance structure provided the best fit for within-subject measurements for all outcomes. For any continuous outcome, if the mean value differed among the treatment groups at baseline (−144 hours), the change from baseline was calculated for all subsequent times and used as the outcome for subsequent modeling. Similar mixed linear models were used to assess neutrophil functionality data (phagocytic and oxidative burst capacities and L-selectin expression) with results also reported for the following 3 preplanned nonorthogonal contrasts: group A versus all 3 other groups at −72 hours, group A versus the mean for group B and the control group combined at 0 hours, and group V versus the mean for group B and the control group combined at 0 hours.
Generalized linear mixed models were used to evaluate the effect of treatment and time on nasal lesion scores; clinical scores for cough, nasal discharge, ocular discharge, and posture; and anti-BHV1 antibody titers. Each model included fixed effects for treatment, time, and the interaction between treatment and time and included a random effect to account for repeated measures within each heifer. For all models, results were reported as the least squares mean ± SEM, and values of P ≤ 0.05 were considered significant. All analyses were performed by use of a commercially available statistical software program.r
Results
Heifers and clinical findings
Thirty-one of 32 heifers completed the study. The interaction between treatment and time was not significant (P ≥ 0.11) for any of the clinical illness scores (cough, nasal discharge, ocular discharge, and posture). The mean scores for cough, nasal discharge, and posture did not differ significantly among the 4 treatment groups at any observation time. The mean score for cough did not change over time (P = 0.60), and only 2 heifers received a cough score ≥ 1 throughout the study. However, the mean scores for posture and nasal discharge did change over time (P < 0.001), with an increase in the proportion of heifers assigned scores ≥ 1 for both variables following BHV1 challenge. The mean ocular discharge score for group V (P = 0.02) was greater than that for each of the other 3 groups and increased over time (P < 0.001).
The least squares mean vaginal temperature for each treatment group was plotted over time (Figure 1). There was an obvious diurnal variation in vaginal temperature for all 4 treatment groups. When data were averaged into 1-hour increments, the mean vaginal temperature increased (P < 0.001) following both the BHV1 challenge and M haemolytica challenge for all 4 treatment groups. The interaction between treatment and time did not affect vaginal temperature (P = 0.67). At M haemolytica challenge (0 hours), the mean vaginal temperature for the control group was greater than that for each of the other 3 treatment groups (P < 0.001). From 0 to 72 hours after M haemolytica challenge, the mean vaginal temperatures for the control group and group A mirrored each other, as did the mean vaginal temperatures for groups B and V. Additionally, the mean vaginal temperatures for the control group and group A were generally numerically greater than those for groups B and V between 0 and 72 hours after M haemolytica challenge but did not differ significantly (P = 0.098) among groups. The mean vaginal temperature for the control group over the duration of the immunologic challenge (−72 to 72 hours) did not differ significantly from that for group A but was greater (P = 0.04) than the mean vaginal temperatures over the duration of the immunologic challenge for groups B and V. Similarly, for the 72 hours after M haemolytica challenge, the mean magnitude of the increase in vaginal temperature from baseline (−144 hour to −72 hours) for the control group did not differ significantly from that for group A but was greater (P = 0.03) than that for both groups B and V.
Least squares mean vaginal temperature over time for beef heifers that were topically administered 1 dose of TDFM (3.3 mg/kg) the day after research center arrival (−144 hours; group A; gray line; n = 8), at the time of BHV1 challenge (−72 hours; group V; red line; 8), or at the time of Mannheimia haemolytica challenge (0 hours; group B; blue line; 8) and that did not receive TDFM (control group; green line; 8). Mannheimia haemolytica challenge inoculation was designated as 0 hours. All 32 heifers received a challenge inoculation of BHV1 (Colorado strain) at −72 hours; 1 mL of inoculum (1 × 108 PFUs of BHV1/mL) was instilled in each nostril by use of a syringe and nasal atomization device. All heifers also received a challenge inoculation with M haemolytica serotype A1 at 0 hours; a mean dose of 1.18 × 106 CFUs of logarithmic-phase M haemolytica was diluted with 50 mL of sterile PBS solution and aseptically injected into the trachea. Each heifer was instrumented with a vaginal temperature-monitoring device from −72 to 72 hours after M haemolytica challenge that recorded temperature every 5 minutes. The data were aggregated into 1-hour intervals, and the mean vaginal temperature was calculated for each 1-hour interval for analysis purposes. Values were determined from a mixed linear model that included fixed effects for treatment, time, and the interaction between treatment and time and a random effect to account for repeated measures within heifers. The SEM for the model was 0.07°C.
Citation: American Journal of Veterinary Research 81, 3; 10.2460/ajvr.81.3.243
Least squares mean vaginal temperature over time for beef heifers that were topically administered 1 dose of TDFM (3.3 mg/kg) the day after research center arrival (−144 hours; group A; gray line; n = 8), at the time of BHV1 challenge (−72 hours; group V; red line; 8), or at the time of Mannheimia haemolytica challenge (0 hours; group B; blue line; 8) and that did not receive TDFM (control group; green line; 8). Mannheimia haemolytica challenge inoculation was designated as 0 hours. All 32 heifers received a challenge inoculation of BHV1 (Colorado strain) at −72 hours; 1 mL of inoculum (1 × 108 PFUs of BHV1/mL) was instilled in each nostril by use of a syringe and nasal atomization device. All heifers also received a challenge inoculation with M haemolytica serotype A1 at 0 hours; a mean dose of 1.18 × 106 CFUs of logarithmic-phase M haemolytica was diluted with 50 mL of sterile PBS solution and aseptically injected into the trachea. Each heifer was instrumented with a vaginal temperature-monitoring device from −72 to 72 hours after M haemolytica challenge that recorded temperature every 5 minutes. The data were aggregated into 1-hour intervals, and the mean vaginal temperature was calculated for each 1-hour interval for analysis purposes. Values were determined from a mixed linear model that included fixed effects for treatment, time, and the interaction between treatment and time and a random effect to account for repeated measures within heifers. The SEM for the model was 0.07°C.
Citation: American Journal of Veterinary Research 81, 3; 10.2460/ajvr.81.3.243
Least squares mean vaginal temperature over time for beef heifers that were topically administered 1 dose of TDFM (3.3 mg/kg) the day after research center arrival (−144 hours; group A; gray line; n = 8), at the time of BHV1 challenge (−72 hours; group V; red line; 8), or at the time of Mannheimia haemolytica challenge (0 hours; group B; blue line; 8) and that did not receive TDFM (control group; green line; 8). Mannheimia haemolytica challenge inoculation was designated as 0 hours. All 32 heifers received a challenge inoculation of BHV1 (Colorado strain) at −72 hours; 1 mL of inoculum (1 × 108 PFUs of BHV1/mL) was instilled in each nostril by use of a syringe and nasal atomization device. All heifers also received a challenge inoculation with M haemolytica serotype A1 at 0 hours; a mean dose of 1.18 × 106 CFUs of logarithmic-phase M haemolytica was diluted with 50 mL of sterile PBS solution and aseptically injected into the trachea. Each heifer was instrumented with a vaginal temperature-monitoring device from −72 to 72 hours after M haemolytica challenge that recorded temperature every 5 minutes. The data were aggregated into 1-hour intervals, and the mean vaginal temperature was calculated for each 1-hour interval for analysis purposes. Values were determined from a mixed linear model that included fixed effects for treatment, time, and the interaction between treatment and time and a random effect to account for repeated measures within heifers. The SEM for the model was 0.07°C.
Citation: American Journal of Veterinary Research 81, 3; 10.2460/ajvr.81.3.243
Water intake during the 72 hours after M haemolytica challenge was affected by treatment (P = 0.007) and time (P < 0.001) but not the interaction between treatment and time (P = 0.427; Table 1). Water intake following M haemolytica challenge was consistently lowest for the control group.
Mixed linear regression results for hematologic variables and water intake for beef heifers that were topically administered 1 dose of TDFM (3.3 mg/kg) the day after research center arrival (−144 hours; group A; n = 8), at the time of BHV1 challenge (−72 hours; group V; 8), or at the time of Mannheimia haemolytica challenge (0 hours; group B; 8) and that did not receive TDFM (control group; 8).
| Treatment group | P values for fixed effects | |||||||
|---|---|---|---|---|---|---|---|---|
| Variable | A | V | B | Control | SEM | Treatment | Time | Interaction between treatment and time |
| Leukocytes (× 103 cells/μL) | 14.84 | 13.85 | 12.39 | 14.74 | 0.619 | 0.361 | < 0.001 | 0.483 |
| Neutrophils (× 103 cells/μL) | 4.23 | 3.87 | 3.62 | 5.29 | 0.382 | 0.743 | < 0.001 | 0.693 |
| Lymphocytes (× 103 cells/μL) | 7.23 | 6.59 | 5.82 | 6.25 | 0.318 | 0.153 | 0.045 | 0.994 |
| Neutrophil-to-lymphocyte ratio | 0.614 | 0.957 | 0.655 | 0.682 | 0.072 | 0.350 | < 0.001 | 0.657 |
| Monocytes (× 103 cells/μL) | 3.48 | 3.40 | 2.28 | 3.19 | 0.218 | 0.157 | < 0.001 | 0.862 |
| Eosinophils (× 103 cells/μL) | 0.052 | 0.059 | 0.106 | 0.066 | 0.0458 | 0.826 | 0.008 | 0.525 |
| Basophils (× 103 cells/μL) | 0.004 | 0.003 | 0.002 | 0.005 | 0.0008 | 0.058 | 0.438 | 0.779 |
| Platelets (× 103 platelets/μL) | 674 | 759 | 703 | 757 | 58.64 | 0.682 | 0.002 | 0.884 |
| RBC count (× 106 cells/μL) | 8.89 | 9.01 | 9.46 | 9.06 | 0.236 | 0.337 | < 0.001 | 0.553 |
| Hemoglobin (g/dL) | 11.16 | 10.95 | 11.04 | 11.25 | 0.305 | 0.897 | < 0.001 | 0.474 |
| Hct (%) | 34.16 | 32.49 | 34.19 | 32.51 | 1.087 | 0.466 | < 0.001 | 0.485 |
| Water intake (mL/h) | 548 | 499 | 486 | 342 | 39.93 | 0.007 | < 0.001 | 0.427 |
Values represent the least squares mean unless otherwise specified. Mannheimia haemolytica challenge inoculation was designated as 0 hours. All 32 heifers received a challenge inoculation of BHV1 (Colorado strain) at −72 hours; 1 mL of inoculum (1 × 108 PFUs of BHV1/mL) was instilled in each nostril by use of a syringe and nasal atomization device. All heifers also received a challenge inoculation with M haemolytica serotype AI at 0 hours; a mean dose of 1.18 × 106 CFUs of logarithmic-phase M haemolytica was diluted with 50 mL of sterile PBS solution and aseptically injected into the trachea. For each heifer, a blood sample for a CBC was obtained from the jugular vein at −144, −72, 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, and 72 hours after M haemolytica challenge. Water intake was monitored only from 0 to 72 hours after M haemolytica challenge. Each model included fixed effects for treatment group (treatment), sample acquisition time (time), and the interaction between treatment and time and a random effect to account for repeated measures within heifers.
Seven of the 8 heifers within each treatment group were seronegative for antibodies against BHV1 at −144 hours; the anti-BHV antibody titer ranged from 4 to 64 for the remaining 4 heifers (1 heifer in each of the 4 groups). However, by 360 hours (18 days after BHV1 challenge), all but 4 heifers had detectable serum anti-BHV1 antibody titers, which ranged from 4 to 256. The mean serum anti-BHV1 antibody titer did not differ (P = 0.079) among the 4 treatments groups at any time but did increase (P < 0.001) for all groups after BHV1 challenge (Figure 2). Similarly, nasal lesion scores did not differ (P = 0.91) among the 4 treatment groups but did increase (P < 0.001) after BHV1 challenge.
Least squares mean serum anti-BHV1 antibody titers for the beef heifers in the control group (green bars) and groups A (gray bars), B (blue bars), and V (red bars) described in Figure 1 at baseline (−144 hours) and 360 hours after M haemolytica challenge. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 81, 3; 10.2460/ajvr.81.3.243
Least squares mean serum anti-BHV1 antibody titers for the beef heifers in the control group (green bars) and groups A (gray bars), B (blue bars), and V (red bars) described in Figure 1 at baseline (−144 hours) and 360 hours after M haemolytica challenge. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 81, 3; 10.2460/ajvr.81.3.243
Least squares mean serum anti-BHV1 antibody titers for the beef heifers in the control group (green bars) and groups A (gray bars), B (blue bars), and V (red bars) described in Figure 1 at baseline (−144 hours) and 360 hours after M haemolytica challenge. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 81, 3; 10.2460/ajvr.81.3.243
Immunologic and metabolic responses
Mixed linear regression results for hematologic variables were summarized (Table 1). Neither treatment nor the interaction between treatment and time had a significant effect on any of the hematologic variables evaluated. Time had a significant effect on all hematologic variables evaluated except basophil count. The mean leukocyte count increased (P < 0.001) following M haemolytica challenge for all 4 treatment groups, and the magnitude of the increase from baseline was similar for both neutrophils and lymphocytes in all 4 groups.
At the time of M haemolytica challenge, the respective mean neutrophil oxidative burst capacities for groups A and V were lower (P = 0.005) than the combined mean neutrophil oxidative burst capacity for group B and the control group (Figure 3). However, the mean neutrophil oxidative burst capacity did not differ significantly among the 4 treatment groups at any of the other sample acquisition times. Mean neutrophil phagocytic capacity did not differ significantly among the 4 treatment groups at any time, although the mean phagocytic capacity for group A at M haemolytica challenge was numerically, albeit not significantly (P = 0.07), lower than that for group B and the control group. The change in neutrophil L-selection expression from baseline was affected (P = 0.004) by the interaction between treatment and time such that the mean change in neutrophil L-selectin expression from baseline was lowest for group V at 0 hours and for group B at 72 hours after M haemolytica challenge.
Least squares mean ± SEM neutrophil oxidative burst capacity (A) and change in neutrophil surface L-selectin expression from baseline (−144 hours; B) for the beef heifers of Figure 1. In panel A, notice that the respective neutrophil oxidative burst capacities for groups A and V were significantly lower than the combined mean neutrophil oxidative burst capacity for group B and the control group at the time of M haemolytica challenge (0 hours). In panel B, a significant (P = 0.004) interaction between treatment and time is evident whereby the mean change in neutrophil L-selectin expression was lowest for group V at 0 hours and for group B at 72 hours. GMFI = Geometric mean fluorescence intensity. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 81, 3; 10.2460/ajvr.81.3.243
Least squares mean ± SEM neutrophil oxidative burst capacity (A) and change in neutrophil surface L-selectin expression from baseline (−144 hours; B) for the beef heifers of Figure 1. In panel A, notice that the respective neutrophil oxidative burst capacities for groups A and V were significantly lower than the combined mean neutrophil oxidative burst capacity for group B and the control group at the time of M haemolytica challenge (0 hours). In panel B, a significant (P = 0.004) interaction between treatment and time is evident whereby the mean change in neutrophil L-selectin expression was lowest for group V at 0 hours and for group B at 72 hours. GMFI = Geometric mean fluorescence intensity. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 81, 3; 10.2460/ajvr.81.3.243
Least squares mean ± SEM neutrophil oxidative burst capacity (A) and change in neutrophil surface L-selectin expression from baseline (−144 hours; B) for the beef heifers of Figure 1. In panel A, notice that the respective neutrophil oxidative burst capacities for groups A and V were significantly lower than the combined mean neutrophil oxidative burst capacity for group B and the control group at the time of M haemolytica challenge (0 hours). In panel B, a significant (P = 0.004) interaction between treatment and time is evident whereby the mean change in neutrophil L-selectin expression was lowest for group V at 0 hours and for group B at 72 hours. GMFI = Geometric mean fluorescence intensity. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 81, 3; 10.2460/ajvr.81.3.243
Mixed linear regression results for select serum cytokines and metabolic biomarkers were summarized (Table 2). Neither treatment nor the interaction between treatment and time had a significant effect on any of the serum variables evaluated, whereas time had a significant effect on all serum variables evaluated. For all 4 treatment groups, mean serum IL-1β and IL-6 concentrations increased following M haemolytica challenge. Serum IFNγ concentration increased following BHV1 challenge, and serum IL-4 concentration increased following M haemolytica challenge.
Mixed linear regression results for select serum cytokines and metabolic biomarkers for the beef heifers of Table 1.
| Treatment group | P values for fixed effects | |||||||
|---|---|---|---|---|---|---|---|---|
| Variable | A | V | B | Control | SEM | Treatment | Time | Interaction between treatment and time |
| IL-1β (pg/mL) | 2.12 | 1.34 | 2.16 | 2.21 | 1.79 | 0.981 | < 0.001 | 0.920 |
| IL-6 (pg/mL) | 126.19 | 77.26 | 85.13 | 91.33 | 26.522 | 0.583 | < 0.001 | 0.692 |
| IFNγ (pg/mL) | 7.43 | 4.18 | 3.26 | 3.78 | 1.482 | 0.208 | < 0.001 | 0.630 |
| IL-4 (pg/mL) | 4.08 | 4.60 | 4.92 | 2.86 | 0.937 | 0.435 | < 0.001 | 0.080 |
| Haptoglobin (μg/dL) | 35,208 | 25,749 | 27,261 | 30,551 | 30.55 | 0.293 | < 0.001 | 0.811 |
| Cortisol (ng/mL) | 22.65 | 22.54 | 23.92 | 21.74 | 1.4811 | 0.775 | < 0.001 | 0.057 |
| Glucose (mg/dL) | 98.22 | 97.93 | 93.41 | 93.40 | 4.148 | 0.765 | < 0.001 | 0.828 |
| NEFA (mEq/L) | 0.297 | 0.279 | 0.251 | 0.276 | 0.0203 | 0.457 | < 0.001 | 0.311 |
| SUN (mg/dL) | 12.99 | 11.62 | 11.94 | 12.53 | 0.449 | 0.160 | < 0.001 | 0.574 |
For each heifer, a blood sample for the acquisition of serum to evaluate the listed variables was obtained from the jugular vein at −144, −72, 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, 72, and 144 hours after M haemolytica challenge.
See Table 1 for remainder of key.
For all 4 treatment groups, serum cortisol concentration was greatest prior to M haemolytica challenge and decreased significantly after the heifers were moved to individual stalls during the period from 0 to 72 hours after M haemolytica challenge. Conversely, serum haptoglobin concentration increased significantly for all 4 treatment groups after M haemolytica challenge.
All 4 treatment groups had similar patterns over time in regard to changes in serum glucose, NEFA, and SUN concentrations. The serum glucose concentration decreased between 0 and 7 hours and then increased between 7 and 8 hours after M haemolytica challenge. The serum NEFA concentration increased between 0 and 4 hours, followed by a transient decrease between 4 and 72 hours after M haemolytica challenge. The SUN concentration increased between 0 and 2 hours and then decreased between 3 and 72 hours after M haemolytica challenge.
Discussion
In the present study, topical application of TDFM effectively controlled pyrexia in beef heifers when it was administered at the same time as a challenge inoculation of BHV1 or M haemolytica without adversely affecting the acute immunologic or metabolic responses of the animals to the pathogen challenge. Administration of TDFM to the heifers 72 hours prior to BHV1 or M haemolytica challenge did not prevent challenge-induced pyrexia.
The BHV1-M haemolytica challenge model used for the heifers of the present study was the same as that used to successfully induce pyrexia and BRD in beef heifers of another study.15 The goal of the present study was to determine whether the timing of TDFM administration relative to respiratory pathogen challenge affected the animals’ acute and metabolic responses to the challenge. Administration of flunixin meglumine was warranted because results of the previous study15 suggested that all heifers would develop pyrexia in response to the challenge inoculations.
For all heifers of the present study, the vaginal temperature exceeded 40°C, the proposed threshold for diagnosis of BRD,16,17 following BHV1 challenge. In general, following the BHV1 challenge, the mean vaginal temperature for the control group was similar to that for group A and greater than the mean vaginal temperature for groups B and V, which suggested that TDFM administration was effective in alleviating BHV1-induced pyrexia when administered at the same time as the challenge but not when administered 72 hours before the challenge. At the time of the M haemolytica challenge (prior to TDFM administration to group B), the mean vaginal temperature for group B was significantly lower than that for the control group. Therefore, the change in vaginal temperature relative to baseline (the mean vaginal temperature from −144 to −72 hours [before any immunologic challenge]) was calculated for each heifer in group B and the control group, and the mean change in vaginal temperature from baseline was compared between those 2 treatment groups. Results indicated that the mean change in vaginal temperature from baseline for group B was significantly greater than that for the control group, which again suggested that TDFM administration effectively controlled M haemolytica-induced pyrexia.
For cattle with BRD, rapid alleviation of pyrexia is the most consistently observed response following NSAID administration.7,18 In a clinical efficacy experiment for TDFM, which was conducted at 4 research sites and involved 235 beef cattle with naturally occurring BRD, the proportion of cattle with a decrease in rectal temperature for 6 hours after TDFM administration was significantly greater for the TDFM-treated group (70/120 [58.3%]) than for an untreated control group (7/115 [6.1%]).4 In another study,19 the mean rectal temperature for Holstein calves with BRD that received tulathromycin and flunixin meglumine (2.2 mg/kg, IV) was significantly lower than that for similar calves that received tulathromycin alone at 24 hours, but not at 48 hours, after flunixin meglumine administration. Crossbred beef calves (mean body weight, 197 kg) with naturally occurring BRD that received an NSAID (flunixin, ketoprofen, or carprofen) in conjunction with ceftiofur had a decrease in rectal temperature for 6 hours after NSAID administration, compared with similar calves that received ceftiofur only; however, rectal temperature did not differ significantly between calves that did and did not receive an NSAID at 48 hours after NSAID administration.20 In yet another experimental study,8 the rectal temperature of cattle that received flunixin meglumine PO or IV 1.5 hours before an endotoxin challenge was significantly lower for 8 hours after flunixin meglumine administration, compared with that for control cattle that did not receive flunixin meglumine before endotoxin challenge. Thus, in the present study, the response in body temperature observed for the heifers of groups B and V following TDFM administration was similar to that of other cattle following flunixin meglumine administration regardless of the route of administration (IV, PO, or topical).
Administration of TDFM to the heifers of group A at 72 hours before BHV1 challenge might have been too early to alleviate BHV1-induced pyrexia. In cattle, a viral infection of the respiratory tract, such as that caused by BHV1, is often a predisposing factor for secondary bacterial infections.21 In the present study, an interval of 72 hours between the BHV1 and M haemolytica challenges was chosen so that BHV1 replication would be at its peak at the time of or shortly after the M haemolytica challenge.22 We believed that administration of TDFM to 1 group of cattle at 72-hour intervals would allow sufficient time for immunologic and metabolic responses specific to a certain event (research center arrival, BHV1 challenge, and M haemolytica challenge) and the effect of TDFM on those responses to be observed or detected.
Prostaglandin E2 is an important mediator of fever,23 and NSAIDs limit PGE2 production by inhibiting cyclooxygenase activity.24 Results of an experimental study5 indicate that administration of TDFM to cattle effectively reduces PGE2 synthesis in inflamed tissues for up to 48 hours after experimental induction of inflammation, with peak PGE2 inhibition occurring 8 hours after TDFM administration. The reported mean half-life of TDFM in cattle is 6.42 hours,6 which may be the reason that research regarding the efficacy of the drug on pyrexia has been limited to 6 hours after administration. Proinflammatory cytokines, such as IL-6 and IL-1β, are associated with rapid induction of pyrexia during infection,25,26 and IL-1β is associated with an increase in PGE2 synthesis in experimental models of infection in murine species.25,27 Although PGE2 concentration was not measured for the heifers of the present study, IL-6 and IL-1β concentrations were measured and did not differ significantly among the 4 treatment groups. That finding suggested that alleviation of pyrexia in groups V and B occurred through biological mechanisms not associated with systemic production of proinflammatory cytokines.
In the present study, water intake during the 72 hours after M haemolytica challenge was significantly lower for the control group, compared with the other 3 treatment groups. Results of other studies indicate that water intake frequency and duration may be similar28 or increased29 in cattle with BRD relative to healthy cattle; however, cattle that are ill may be less willing to seek out water.30 In another study,31 Holstein calves with naturally occurring neonatal diarrhea that were treated with the NSAID meloxicam had increased water and starter ration intake, compared with placebo-treated calves, and the authors attributed the increase in water intake to a decrease in sickness-related behavior and an increase in appetite. Although alleviation of M haemolytica-induced pyrexia by TDFM might have made the heifers of group B feel better and thereby increase their water consumption, it does not explain why water intake during the 72 hours after the M haemolytica challenge was greatest for the heifers of group A and lowest for the heifers of the control group, especially given that the mean vaginal temperature for group A mirrored that of the control group during that period. Further research is necessary to elucidate the effects of NSAIDs on the water intake of diseased cattle.
The leukocyte, neutrophil, and lymphocyte counts for the heifers of the present study did not appear to be affected by TDFM administration. However, transient decreases in the oxidative burst and phagocytic capacities of neutrophils and surface expression of L-selectin on neutrophils were observed immediately after administration of TDFM, which might indicate that TDFM administration before or during a BRD challenge may impair neutrophil function and thereby decrease neutrophil-associated inflammation. In another study,8 heifers administered flunixin by the PO or IV route 1.5 hours before an endotoxin challenge had no changes in circulating neutrophil or mononuclear cell counts for 7 hours after endotoxin challenge. Flunixin meglumine is associated with inhibition of neutrophil chemotaxis32 and a decrease in lymphocyte proliferation33 in bovine blood in vitro. However, in vitro exposure of blood obtained from neonatal calves to flunixin meglumine causes a slight increase in neutrophil migration.34 An important pathophysiologic consequence of BRD is the potential for leukocyte-induced inflammation to cause lung tissue damage or consolidation.35 Results of 2 studies20,36 indicate that the extent of lung consolidation in beef cattle with BRD was significantly less when the cattle were treated with flunixin meglumine in conjunction with an antimicrobial, compared with when cattle were treated with an antimicrobial alone, whereas results of another study37 indicate no difference in the extent of lung consolidation between cattle with BRD that were and were not treated with flunixin meglumine in conjunction with an antimicrobial. A decrease in lung consolidation is plausible if flunixin meglumine truly impairs the inflammatory activity of neutrophils in cattle with BRD.
In the present study, the mean cortisol concentration during the 144 hours after M haemolytica challenge did not differ significantly among the 4 treatment groups, which suggested that TDFM had no effect on serum cortisol concentration. This was in contrast to results of another study8 in which the mean plasma cortisol concentration for heifers that received flunixin meglumine by the PO or IV route 1.5 hours before an endotoxin challenge was significantly lower than that for control heifers at 75 minutes after the endotoxin challenge; however, the plasma cortisol concentration did not differ significantly between flunixin meglumine-treated and control heifers at any other evaluated time. Results of another study38 suggest that there is a tendency for plasma cortisol concentration to be decreased following transport when beef cattle are administered flunixin meglumine by the IV route immediately before and after 24 hours of transport. Discrepancies regarding the effect of flunixin meglumine on the cortisol concentration between those studies8,38 and the present study might be attributable to differences in the challenge models. Alternatively, cortisol can be synthesized in response to inflammation, and the lack of a significant difference in cortisol concentration among the 4 treatment groups of the present study might have been a reflection of a lack of significant difference in the proinflammatory cytokine IL-1β concentration among the 4 treatment groups.
For the heifers of the present study, glucose, NEFA, and SUN concentrations were unaffected by TDFM administration. Results of a study39 involving neonatal dairy calves that underwent an endotoxin challenge likewise indicate that administration of flunixin meglumine had no effect on serum glucose concentration when it was administered at the time of the endotoxin challenge. As previously stated, NSAIDs such as flunixin meglumine inhibit cyclooxygenase, which impairs PGE2 synthesis. Prostaglandin E2 induces muscle protein catabolism in vitro,40 and if the same holds true in vivo, synthesis of PGE2 could lead to an increase in SUN. Results of 1 study38 indicate that serum NEFA concentrations in cattle remain increased for 4 days when flunixin meglumine is administered before and after transport, although the reason for that increase remains unknown. Conversely, administration of flunixin meglumine had no apparent effect on the serum NEFA concentrations of the cattle of the present study and other studies.41,42 Collectively, the results of the present study suggested that TDFM administration to cattle had no effect on adipose or muscle catabolism for 144 hours after completion of the BHV1-M haemolytica challenge.
Results of the present study indicated that TDFM administration to beef cattle effectively controlled pyrexia associated with BHV1 and M haemolytica challenges when the drug was administered at the same time as the challenge inoculation; however, TDFM did not alleviate pyrexia when it was administered 72 hours before the BHV1 or M haemolytica challenge. Additionally, TDFM administration did not affect most acute immunologic or metabolic responses of the cattle to the BHV1 and M haemolytica challenges, although it did impair neutrophil functionality, which could decrease neutrophil-associated inflammation. Consequently, administration of TDFM to beef cattle with BRD appeared to be effective for alleviation of pyrexia without any adverse effects on the acute immunologic and metabolic responses to the respiratory pathogens.
Acknowledgments
This study was conducted at the Livestock Issues Research Unit Animal Research Complex, Lubbock, Tex.
This manuscript represents a portion of a dissertation submitted by Dr. Word to the graduate faculty of Texas Tech University in partial fulfillment of the requirements for a Doctor of Philosophy degree.
Partially funded by Merck Animal Health, Summit, NJ. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.
The USDA prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and, where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc) should contact the USDA's TARGET Center at (202) 720–2600 (voice and telecommunications device for the deaf). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Ave, SW, Washington, DC 20250, or call (800) 795–3272 (voice) or (202) 720–6382 (telecommunications device for the deaf). The USDA is an equal opportunity provider and employer.
The authors thank Jeff W. Dailey, Jessica R. Carroll, and Ryan Buchanan for technical assistance.
ABBREVIATIONS
| BHV1 | Bovine herpesvirus I |
| BRD | Bovine respiratory disease |
| IFNγ | Interferon γ |
| IL | Interleukin |
| NEFA | Nonesterified fatty acid |
| PFU | Plaque-forming unit |
| PGE2 | Prostaglandin E2 |
| TDFM | Transdermal flunixin meglumine |
Footnotes
Inforce 3, Zoetis, Parsippany, NJ.
Bovi-Shield Gold 5, Zoetis, Parsippany, NJ.
Purina Animal Nutrition, Gray Summit, Mo.
Banamine Transdermal, Merck Animal Health, Summit, NJ.
Teleflex, Morrisville, NC.
Zuprevo, Merck Animal Health, Summit, NJ.
Suevia Cup, QC Supply Inc, Schuyler, Neb.
Vacutainer, Becton, Dickinson, and Co, Franklin Lakes, NJ.
Sarstedt Inc, Newton, NC.
Idexx Laboratories, Westbrook, Me.
Accuri flow cytometer, Becton, Dickinson, and Co, Franklin Lakes, NJ.
Monoclonal antibody IgG1-isotype in the mouse, VMRD Inc, Pullman, Wash.
Arbor Assays, Ann Arbor, Mich.
Immunology Consultants Laboratory Inc, Portland, Ore.
Wako Pure Chemical Industries, Chuo-Ku, Osaka, Japan.
Wako Diagnostics, Richmond, Va.
Searchlight-Quanterix Inc, Billerica, Mass.
SAS, version 9.417, SAS Institute Inc, Cary, NC.
References
1. USDA APHIS. Types and costs of respiratory disease treatments in US feedlots. Available at: www.aphis.usda.gov/animal_health/nahms/feedlot/downloads/feedlot2011/Feed11_is_RespDis.pdf. Accessed Sep 4, 2019.
2. Pyörälä S, Laurila T, Lehtonen S, et al. Local tissue damage in cows after intramuscular administration of preparations containing phenylbutazone, flunixin, ketoprofen, and metamizole. Acta Vet Scand 1999;40:145–150.
3. USDA Food Safety Inspection Service. United States National Residue Program for Meat, Poultry, and Egg Products: FY 2016 residue sample results. Available at: www.fsis.usda.gov/wps/wcm/connect/d84a5cac-5b4e-4e60-85b4-8886d0dc1660/2016-Red-Book.pdf?MOD=AJPERES. Accessed Aug 29, 2017.
4. FDA. Freedom of information summary, original new animal drug application 141–450, banamine transdermal (flunixin transdermal solution). Available at: animaldrugsatfda.fda.gov/adafda/app/search/public/document/downloadFoi/1944. Accessed Sep 4, 2019.
5. Thiry J, Fournier R, Roy O, et al. Evaluation of flunixin meglumine pour-on administration on prostaglandin E2 concentration in inflammatory exudate after induction of inflammation in cattle. Res Vet Sci 2017;114:294–296.
6. Kleinhenz MD, Van Engen NK, Gorden PJ, et al. The pharmacokinetics of transdermal flunixin meglumine in Holstein calves. J Vet Pharmacol Ther 2016;39:612–615.
7. Apley M. Ancillary therapy of bovine respiratory disease. Vet Clin North Am Food Anim Pract 1997;13:575–592.
8. Odensvik K, Magnusson U. Effect of oral administration of flunixin meglumine on the inflammatory response to endotoxin in heifers. Am J Vet Res 1996;57:201–204.
9. Bednarek D, Zdzisizska B, Kondracki M, et al. A comparative study of the effects of meloxicam and flunixin meglumine (NSAIDs) as adjunctive therapy on interferon and tumor necrosis factor production in calves suffering from enzootic bronchopneumonia. Pol J Vet Sci 2003;6:109–115.
10. Glynn HD, Coetzee JF, Edwards-Callaway LN, et al. The pharmacokinetics and effects of meloxicam, gabapentin, and flunixin in postweaning dairy calves following dehorning with local anesthesia. J Vet Pharmacol Ther 2013;36:550–561.
11. Federation of Animal Science Societies. Guide for care and use of agricultural animals in research and teaching. 3rd ed. Champaign, Ill: FASS Inc, 2010.
12. Burdick NC, Carroll JA, Dailey JW, et al. Development of a self-contained, indwelling vaginal temperature probe for use in cattle research. J Therm Biol 2012;37:339–343.
13. School of Veterinary Medicine, University of Wisconsin-Madison. Calf respiratory scoring chart. Available at: www.vetmed.wisc.edu/dms/fapm/fapmtools/8calf/calf_respiratory_scoring_chart.pdf. Accessed Jul 21, 2017.
14. Obeidat BS, Cobb CJ, Sellers MD, et al. Plane of nutrition during the preweaning period but not the grower phase influences the neutrophil activity of Holstein calves. J Dairy Sci 2013;96:7155–7166.
15. Word AB, Broadway PR, Burdick Sanchez NC, et al. Immune and metabolic responses of beef heifers supplemented with Saccharomyces cerevisiae to a combined viral-bacterial disease challenge. Transl Anim Sci 2019;3:135–148.
16. Perino LJ, Apley MD. Clinical trial design in feedlots. Vet Clin North Am Food Anim Pract 1998;14:343–365.
17. Duff GC, Galyean ML. Board-invited review: recent advances in management of highly stressed, newly received feedlot cattle. J Anim Sci 2007;85:823–840.
18. Francoz D, Buczinski S, Apley M. Evidence related to the use of ancillary drugs in bovine respiratory disease (anti-inflammatory and others): are they justified or not? Vet Clin North Am Food Anim Pract 2012;28:23–38.
19. Guzel M, Karakurum MC, Durgut R, et al. Clinical efficacy of diclofenac sodium and flunixin meglumine as adjuncts to antibacterial treatment of respiratory disease of calves. Aust Vet J 2010;88:236–239.
20. Lockwood PW, Johnson JC, Katz TL. Clinical efficacy of flunixin, carprofen and ketoprofen as adjuncts to the antibacterial treatment of bovine respiratory disease. Vet Rec 2003;152:392–394.
21. Taylor JD, Fulton RW, Lehenbauer TW, et al. The epidemiology of bovine respiratory disease: what is the evidence for predisposing factors? Can Vet J 2010;51:1095–1102.
22. Jericho KW, Langford EV. Pneumonia in calves produced with aerosols of bovine herpesvirus 1 and Pasteurella haemolytica. Can J Comp Med 1978;42:269–277.
23. Ushikubi F, Segi E, Sugimoto Y, et al. Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP3. Nature 1998;395:281–284.
24. Lees P, Landoni MF, Giraudel J, et al. Pharmacodynamics and pharmacokinetics of nonsteroidal anti-inflammatory drugs in species of veterinary interest. J Vet Pharmacol Ther 2004;27:479–490.
25. Exton MS. Infection-induced anorexia: active host defence strategy. Appetite 1997;29:369–383.
26. Carroll JA, Reuter RR, Chase CC, et al. Profile of the bovine acute-phase response following an intravenous bolus-dose li-popolysaccharide challenge. Innate Immun 2009;15:81–89.
27. Katsuura G, Gottschall PE, Dahl RR, et al. Interleukin-1 beta increases prostaglandin E2 in rat astrocyte cultures: modulatory effect of neuropeptides. Endocrinology 1989;124:3125–3127.
28. Sowell BF, Branine ME, Bowman JG, et al. Feeding and watering behavior of healthy and morbid steers in a commercial feedlot. J Anim Sci 1999;77:1105–1112.
29. Buhman MJ, Perino LJ, Galyean ML, et al. Association between changes in eating and drinking behaviors and respiratory tract disease in newly arrived calves at a feedlot. Am J Vet Res 2000;61:1163–1168.
30. Hart BL. Biological basis of the behavior of sick animals. Neurosci Biobehav Rev 1988;12:123–137.
31. Todd CG, Millman ST, McKnight DR, et al. Nonsteroidal antiinflammatory drug therapy for neonatal calf diarrhea complex: effects on calf performance. J Anim Sci 2010;88:2019–2028.
32. Wernicki A, Urban-Chmiel R, Puchalski A, et al. Evaluation of the in vitro effect of different concentrations of flunixin on leukocytes obtained from cattle of various ages. Vet Zootechnika 2015;72:37–44.
33. Maeda Y, Tanaka R, Ohtsuka H, et al. Comparison of the immunosuppressive effects of dexamethasone, flunixin meglumine and meloxicam on the in vitro response of calf peripheral blood mononuclear cells. J Vet Med Sci 2011;73:957–960.
34. Zwahlen RD, Roth DR. Chemotactic competence of neutrophils from neonatal calves. Functional comparison with neutrophils from adult cattle. Inflammation 1990;14:109–123.
35. Gardner BA, Dolezal HG, Bryant LK, et al. Health of finishing steers: effects on performance, carcass traits, and meat tenderness. J Anim Sci 1999;77:3168–3175.
36. Friton GM, Cajal C, Ramirez-Ramero R. Long-term effects of meloxicam in the treatment of respiratory disease in fattening cattle. Vet Rec 2005;156:809–811.
37. Wilson BK, Step DL, Maxwell CL, et al. Evaluation of multiple ancillary therapies used in combination with an antimicrobial in newly received high-risk calves treated for bovine respiratory disease. J Anim Sci 2015;93:3661–3674.
38. Cooke RF, Cappellozza BI, Guarnieri Filho TA, et al. Effects of flunixin meglumine administration on physiological and performance responses of transported feeder cattle. J Anim Sci 2013;91:5500–5506.
39. Semrad SD. Comparison of flunixin, prednisolone, dimethyl sulfoxide, and a lazaroid (U74389F) for treating endotoxemic neonatal calves. Am J Vet Res 1993;54:1517–1522.
40. Baracos V, Rodemann HP, Dinarello CA, et al. Stimulation of muscle protein degradation and prostaglandin E2 release by leukocytic pyrogen (interleukin-1). A mechanism for the increased degradation of muscle proteins during fever. N Engl J Med 1983;308:553–558.
41. Shwartz G, Hill KL, VanBaale MJ, et al. Effects of flunixin meglumine on pyrexia and bioenergetic variables in postparturient dairy cows. J Dairy Sci 2009;92:1963–1970.
42. Giammarco M, Fusaro I, Vignola G, et al. Effects of a single injection of flunixin meglumine or carprofen postpartum on haemotological parameters, productive performance and fertility of dairy cattle. Anim Prod Sci 2018;58:322–331.
Appendix
Description of the clinical illness scoring system used to subjectively assess 32 beef crossbred heifers that did or did not receive topical application of TDFM (3.3 mg/kg) at various times relative to experimental inoculation with BHV1 and Mannheimia haemolytica.
| Score | ||||
|---|---|---|---|---|
| Variable | 0 | 1 | 2 | 3 |
| Cough | None | Single induced cough | Repeated induced cough or single spontaneous cough | Repeated spontaneous coughs |
| Nasal discharge | Normal serous discharge | Small amount of cloudy discharge unilaterally | Cloudy or excessive mucous discharge bilaterally | Copious mucopurulent discharge bilaterally |
| Ocular discharge | Normal | Small amount of discharge | Moderate amount of discharge bilaterally | Large amount of discharge bilaterally |
| Posture | Normal | Stands with head and neck slightly distended | Stands with head and neck distended and ears drooping | Reluctant to rise |
Adapted from a scoring system12 developed for dairy calves. Each heifer was assigned a clinical illness score on a daily basis at approximately 8 am by 1 of 2 trained personnel throughout the study.
