Modulation of the metabolic response using dexamethasone in beef steers vaccinated with a multivalent respiratory vaccine

Abstract

Available energy plays a critical role in the initiation and maintenance of an immune response to a pathogen, a process that is further altered by activation of the stress system. This study was designed to determine the effect of an acute vs chronic stress model on the metabolic response to vaccination in naïve beef steers. Steers (n = 32; 209 ± 8 kg) were blocked by body weight (BW) and randomly assigned to one of three treatments: 1) Chronic stress (CHR), 0.5 mg/kg BW dexamethasone (DEX) administered i.v. at 1000 h on day 3 to day 0; 2) Acute stress (ACU), 0.5 mg/kg BW DEX administered i.v. at 1000 h on day 0 only; or 3) Control (CON), no DEX. On day −4, steers were fitted with jugular vein catheters and moved into individual bleeding stalls in an environmentally-controlled facility. Blood samples were collected at −74, −50, and −26 h, at 0.5-h intervals from −4 to 6 h, and at 12, 24, 36, 48, and 72 h relative to vaccination with a combination vaccine (Pyramid 5 + Presponse SQ, Boehringer Ingelheim Animal Health USA, Duluth, GA) at 1200 h on day 0. Data were analyzed by the MIXED procedure of SAS specific for repeated measures. There was a treatment × time interaction (P < 0.001) for serum glucose concentrations. Specifically, glucose concentrations increased at −50 h in CHR steers and at 1200 h in ACU steers and remained elevated through 72 h postvaccination period in these two treatments compared to CON steers. The change in nonesterified fatty acid (NEFA) concentrations relative to baseline values was affected by treatment and time (P < 0.001) such that the change in NEFA was greater in CHR (0.06 ± 0.01 mmol/L), followed by CON (−0.01 ± 0.01 mmol/L) and ACU steers (−0.04 ± 0.01 mmol/L). There was a tendency (P = 0.08) for a treatment × time interaction for change in serum NEFA concentrations. Serum urea nitrogen (SUN) was affected by treatment and time (P < 0.001) such that SUN concentrations were greatest in CHR (12.0 ± 0.1 mg/dL) followed by ACU (10.4 ± 0.1 mg/dL) and CON steers (9.6 ± 0.1 mg/dL); however, the treatment × time interaction was not significant (P = 0.12). These data demonstrate that activation of the stress and immune axes using an acute or chronic stress model can increase energy mobilization prior to and following vaccination in naïve steers, potentially affecting available energy needed to mount an adequate antibody response to vaccination.

INTRODUCTION

Vaccination is a common management procedure used by producers to prevent or reduce the negative effects of various pathogens. According to a 2010 report, almost 70% of beef cattle were vaccinated at least once (United States Animal and Plant Health Inspection Service, Veterinary Services, Centers for Epidemiology and Animal Health, 2010). However, this is dependent on industry segment as only approximately 30% of calves were vaccinated against respiratory diseases prior to weaning yet close to 100% of calves are vaccinated at the feedlot. Additionally, vaccination is often accompanied by other management procedures, including weaning, castration and transportation, which are known stressors (Mackenzie et al., 1997; Buckham Sporer et al., 2008). Transportation exposes cattle to additional stressors including commingling, new environments, and new rations. As stress has been demonstrated to negatively impact immune function and responsiveness, the combination of these stressors may influence vaccine efficacy (Mackenzie et al., 1997). Available energy plays a critical role in the initiation and maintenance of an immune response to a pathogen and can be further altered in response to stress. Specifically, cortisol can stimulate glycogenolysis, gluconeogenesis, and catabolism of adipose and protein resulting in increased circulating glucose and fatty acids (Kyrou and Tsigos, 2009). Further, the duration of stress may influence energy reserves and the manner that energy is redistributed throughout the body (Kyrou and Tsigos, 2009).

Data from our laboratory suggests that exposing cattle to dexamethasone (DEX) prior to vaccination can alter the antibody response to a multivalent respiratory vaccine (Richeson et al., 2016). Specifically, DEX treatment increased the antibody response to the modified-live virus vaccine components but reduced the antibody response to the Mannheimia haemolytica toxoid component. Further, the acute phase response, including complete blood counts, serum haptoglobin and ceruloplasmin concentration, neutrophil functionality, and cytokine production were altered by DEX administration (Richeson et al., 2016; Hughes et al., 2017). Based on these results, it was hypothesized that DEX may have caused changes in energy redistribution. Thus, this third and final manuscript from the aforementioned study describes the metabolic response of naïve beef steers exposed to DEX prior to vaccination with a multivalent respiratory vaccine.

MATERIALS AND METHODS

This study was conducted from December 2014 to February 2015. The initial 7-day data collection period reported herein was conducted at the USDA-ARS Livestock Issues Research Unit near Lubbock, TX. All experimental procedures were in compliance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching and was approved by the animal care and use committee at the Livestock Issues Research Unit (protocol # 2014-10-JTR20).

Animals and Housing

Thirty-two Angus × Hereford steers were weaned and backgrounded in an isolated pen at their ranch of origin in central New Mexico 24 d prior to their transport to Lubbock, TX for the start of this study. Cattle were weaned on day −29 relative to vaccination, and blood was collected to confirm seronegative status to infectious bovine rhinotracheitis virus (IBRV), bovine viral diarrhea virus (BVDV), parinfluenza-3 virus (PI3V), and bovine respiratory syncytial virus (BRSV). A subset of calves of moderate temperament was selected from a larger group of cattle using a recorded exit velocity on day −29. Five days prior to vaccination, steers were transported to the USDA-ARS Bovine Immunology Research and Development Facility (Lubbock, TX) from the ranch of origin (450 km distance) in a sanitized trailer. Calves rested overnight in dirt pens with ad libitum access to feed and water. Indwelling jugular catheters and rectal temperature (RT) recording devices (Reuter et al., 2010) were placed in steers the following morning (0800 h; day −4) to facilitate i.v. administration of DEX and serial blood collection for analyses. Cattle were placed into individual bleeding stalls (2.28 m in length, 0.76 m in width, and 1.67 m in height) in an enclosed, ventilated barn following placement of indwelling jugular catheters and RT devices. While in the barn, orts were weighed, and calves were fed daily at 1200 h. Calves were fed a standard diet intended for receiving cattle that was supplied by WTAMU. Cattle were removed from the stanchions on day 3, and briefly restrained in a chute for removal of the jugular catheter and RT recording device. The present study ended at this time and steers were transported to the WTAMU Animal Health Research Facility (Canyon, TX) for a second study period reported in a companion manuscript (Richeson et al., 2016).

Treatments and Vaccination

Steers were weighed on day 4 and stratified by BW (average BW = 209 ± 8 kg) prior to random assignment to one of the three experimental treatments. Randomization was achieved through a drawing of labeled cards from a hat, with treatments consisting of 1) Acute stress (ACU) induced by single i.v. administration of 0.5 mg/kg BW DEX at 1000 h on day 0 only; 2) Chronic stress (CHR) induced by repeated i.v. administration of 0.5 mg/kg BW DEX (1000 h on day 3 to day 0); or 3) Control (CON), no DEX administered. Cards drawn from the hat were replaced, redrawn, and treatments assigned according to BW stratification, resulting in 11, 11, and 10 animals assigned to ACU, CHR and CON treatments, respectively. Two steers assigned to CHR died of undetermined causes on day 0 and day 6, respectively. Data from these calves was excluded from the analysis.

On day 0, steers were administered 2 mL of a multivalent combination respiratory vaccine [Pyramid 5 + Presponse SQ, Boehringer Ingelheim Animal Health USA, Duluth, GA] s.c. in the neck at 1200 h (0 h). Antibody responses against IBRV, BVDV type 1 and 2, PI3V, BRSV, and M. haemolytica whole cell wall and leukotoxin and serum and ceruloplasmin and haptoglobin concentrations are published in the companion manuscript (Richeson et al., 2016). Additionally, the acute phase response, including RT, complete blood counts, proinflammatory cytokines, and neutrophil function are published in a second companion manuscript (Hughes et al., 2017).

Blood Collection and Serology

Beginning on day −3, blood was collected via jugular catheter into a 9-mL sampling tube without additive (Sarstedt Inc., Newton, NC) at −74, −50, and −26 h, at 0.5-h intervals from −4 h to 6 h, and at 12, 24, 36, 48, and 72 h relative to vaccination. Samples were allowed to clot at 20°C for 30 min prior to centrifugation at 1,500 × g for 20 min at 4°C. Serum was harvested and stored in triplicate aliquots at −80°C for analysis of serum glucose, nonesterified fatty acid (NEFA), and serum urea nitrogen (SUN) concentrations.

Glucose concentrations were determined by modification of the enzymatic Autokit Glucose (Wako Diagnostics, Richmond, VA) to fit a 96-well format as previously described (Burdick Sanchez et al., 2014). Briefly, 300 µL of prepared working solution was added to 2 µL of serum or prepared standards in a 96-well plate. Plates were incubated at 37^oC for 5 min and absorption was recorded at 505 nm. The plate reader used for this assay (BioTek Powerwave 340; BioTek Instruments, Winooski, VT) has an incubating and timing feature and therefore ensured that the sample absorbances were read immediately following the 5-min incubation. Concentrations of glucose were determined by comparing unknown samples to a standard curve of known glucose concentrations. The intra and interassay coefficients of variation were less than 5.7% and 7.9%, respectively.

Concentrations of NEFAs were determined by modification of the enzymatic HR Series NEFA-HR (2) assay (Wako Diagnostics, Richmond, VA) to fit a 96-well format as previously described (Burdick Sanchez et al., 2014). Briefly, 200 µL of the prepared Color Reagent A were added to 5 µL of serum or prepared standards in a 96-well plate. Plates were incubated at 37^oC for 5 min and then the absorbance was read using a spectrophotometer at 550 nm. Next, 100 µL of prepared Color Reagent B was added to all wells on the 96-well plate. Plates were incubated for an additional 5 min and read for a second time using a plate reader at 550 nm. A final absorbance was obtained by subtracting the first reading, which was multiplied by a factor of 0.67 to account for changes in volume, from the second reading. The final absorbance values were used for all calculations (i.e., standard curve, sample concentrations). Concentrations of NEFAs were determined by comparing unknown samples to a standard curve of known NEFA concentrations. The intra and interassay coefficients of variation were less than 9.6% and 10.2%, respectively.

Concentrations of SUN were determined by a colorimetric assay according to the manufacturer’s directions (K024-H1; Arbor Assays, Ann Arbor, MI) by comparison of unknowns to standard curves generated with known concentrations of urea nitrogen. The intra and interassay coefficients of variation were less than 5.9% and 8.0%, respectively

Statistical Analyses

This study was a randomized incomplete block design with steer within treatment serving as experimental unit. Serum glucose, NEFA, and SUN data were analyzed using the MIXED procedure (SAS, Inc., Cary, NC), specific for repeated measures. Variance Components covariance structure was used based on having the lowest AICC fit statistic value. Treatment, time, and treatment × time were included as fixed effects. When significant, fixed effect means were separated using the PDIFF option in SAS. P < 0.05 was considered significant, and 0.05 ≤ P ≤ 0.10 was considered a tendency. Data are presented as LSM ± SE.

RESULTS AND DISCUSSION

Stress experienced for a short duration of time, or acute stress, can be beneficial in the fact that it can stimulate or prime the immune system (Dhabhar, 2002; Martin, 2009). However, chronic stress, or stress experienced or repeated over an extended duration of time, can have deleterious effects on immune function, leaving an animal more susceptible to pathogenic infections (Shi et al., 2003), and can also impact metabolism (Kyrou and Tsigos, 2009). Our particular interest was the effects of chronic stress, such as the combination of weaning, transportation, and commingling of cattle, on the response to vaccination. While companion manuscripts have outlined the antibody and acute phase immune responses to vaccination in combination with single or repeated DEX administration in naïve beef steers, the effects of vaccination and DEX administration on various metabolic parameters are described herein.

There was a treatment × time interaction for serum glucose concentrations (P < 0.001; Figure 1). Specifically, CHR steers had greater glucose than CON and ACU steers from −50 through 6 h postvaccination. The rise in glucose in the CHR and ACU steers occurred in the blood sample immediately following the first (or only) dose of DEX. Additionally, ACU steers had greater glucose concentrations than CON steers at 5.5 h. Both ACU and CHR steers had greater glucose than CON steers from 12 to 48 h postvaccination. Thus, it appears that there was no effect of vaccination on the serum glucose response, with a greater influence of DEX administration. The repeated DEX treatment in CHR steers continuously elevated serum glucose concentrations throughout the study, a response that was observed in ACU steers beginning at approximately 5.5 h following vaccination, or 7.5 h following DEX administration. Glucose concentrations began to decline following cessation of DEX administration, as observed in the decrease in glucose concentrations following the 24 h sample, and return to values in CHR and ACU steers not different from CON steers by 72 h. The temporal glucose response to DEX administration is similar to a study where 10 mg DEX was administered intramuscularly to lactating dairy cows (Maplesden et al., 1960). Work by Kronfeld and Hartmann (1973), where DEX was also administered to lactating dairy cows, concluded that the increase in glucose concentrations observed in response to DEX was likely not due to production of glucose but rather due to glucose redistribution. This is supported by work in neonatal calves where the authors found no change in enzymes regulating gluconeogenesis in the liver of calves administered DEX, and also found reduced glucose utilization (Hammon et al., 2003). Acute stress typically results in increases in glucose and other metabolites that are important for supplying energy to tissues that require energy for the fight or flight response (i.e., muscles; (Ricart-Jané et al., 2002). However, prolonged release of glucocorticoids can have negative effects on energy metabolism, resulting in insulin resistance, and increased fat accumulation, which can be further stimulated by increased inflammation (Kyrou and Tsigos, 2009). Additionally, DEX has been reported to induce insulin resistance, which may explain the chronic elevation in glucose in the CHR treatment group (Severino et al., 2002).

Figure 1.

Serum glucose concentrations in steers exposed to acute (ACU), chronic (CHR) or no (CON) dexamethasone treatment relative to vaccination with respiratory vaccine in naïve steers at 0 h. Treatment: P < 0.001; Time: P < 0.001; Treatment × time: P < 0.001. ^abcP ≤ 0.05 for treatments within hour.

Prior to administering DEX to any of the steers, there was a treatment effect (P < 0.001) for serum NEFA (0.12, 0.18, and 0.07 ± 0.03 mmol/L for CON, ACU, and CHR, respectively). It is unclear why this treatment difference occurred, considering the steers had been treated similarly and were randomly allotted to their treatments, and is likely an anomaly. Due to the difference in NEFA concentrations prior to treatment application, NEFA concentrations were evaluated based on the change in response based on the −74-h sample, which was collected immediately prior to administration of any treatment. The change in NEFA concentrations was affected by treatment and time (P < 0.001; Figure 2) such that there was a greater change in NEFA concentrations in CHR steers (0.06 ± 0.01 mmol/L), compared to Control (−0.01 ± 0.01 mmol/L), and ACU steers (−0.04 ± 0.01 mmol/L). All three treatments differed (P ≤ 0.03) from one another. Additionally, there was a tendency (P = 0.08) for a treatment × time interaction where CHR steers had greater change in NEFA concentrations than ACU steers at −50, 0.5, 1.5, 2.5 and 5.5 h relative to vaccination. Serum urea nitrogen concentrations were influenced by treatment and time (P < 0.001; Figure 3). Specifically, all treatments differed from one another, with CON having the lowest SUN concentrations, followed by ACU and then CHR with the greatest concentration. There was no treatment × time interaction (P = 0.12) for SUN.

Figure 2.

The change in serum NEFA concentrations in steers exposed to acute (ACU), chronic (CHR) or no (CON) dexamethasone treatment relative to vaccination with respiratory vaccine in naïve steers at 0 h. Treatment: P < 0.001; Time: P < 0.001; Treatment × time: P = 0.08.

Figure 3.

Serum urea nitrogen concentrations in steers exposed to acute (ACU), chronic (CHR) or no (CON) dexamethasone treatment relative to vaccination with respiratory vaccine in naïve steers at 0 h. Treatment: P < 0.001; Time: P < 0.001; Treatment × time: P = 0.12.

As discussed earlier, glucose concentrations rapidly increase in response to stress, due to a release of glucose from glycogen stores as well as a reduction in glucose storage through a reduction in the response to insulin (i.e., increased insulin insensitivity; (Sapolsky et al., 2000). In a similar manner, glucocorticoids can increase lipids and amino acids through stimulation of lipolysis and proteolysis (Sapolsky et al., 2000). In these actions, glucocorticoids work synergistically with other hormones such as catecholamines, growth hormone and glucagon; however, the effects of glucocorticoids on glucose concentrations extend for a longer period of time as compared to the actions of catecholamines and glucagon. These metabolic substrates are utilized by different tissues and organs of the body (e.g., muscle) for energy during periods of stress, but uptake may be reduced in tissues/organs that are not needed by the body to recover from a stressor. There are variable metabolic responses to stressors reported in the literature. A study in rats exposed to chronic immobilization stress observed no differences in glucose concentrations, yet had an increased glucose/insulin ratio, and had greater NEFA concentrations compared to control rats, while acute immobilization stress did not affect NEFA concentrations (Ricart-Jané et al., 2002). Further, rats treated with DEX had increased NEFA concentrations (Dumas et al., 2005). Serum urea nitrogen was increased in neonatal pigs orally administered DEX for 15 d (Weiler et al., 1997). These data support the observations in the present study for NEFA and SUN; however, there are few studies that report differences in these metabolic parameters in livestock species, and therefore, this is an area that requires further study.

While there were no direct effects of vaccination on the metabolic parameters measured in this study, the changes observed in the metabolic parameters in response to DEX administration may have influenced the responses observed to vaccination and the acute phase response reported in companion manuscripts (Richeson et al., 2016; Hughes et al., 2017). An activated immune system has a large energy requirement, particularly for glucose, and thus any changes in energy availability and update may greatly influence the functionality of the immune system (Kvidera et al., 2017). In vitro studies, particularly in rodents, have demonstrated effects of DEX on immune cell metabolism. Specifically, DEX exposure reduced glucose uptake and utilization in macrophages (Norton and Munck, 1980). In neutrophils, exposure to DEX in vitro resulted in increased glucose uptake, but did not influence glucose oxidation, suggesting an increase in glucose accumulation within neutrophils (Garcia et al., 2003). Furthermore, expression of the enzyme glucose-6-phosphate dehydrogenase, an enzyme important in production of the neutrophil oxidative burst, was reduced in DEX treated rats (Garcia et al., 2003). This supports data from this study reported in a companion manuscript where neutrophil oxidative burst was reduced in ACU and CHR steers (Hughes et al., 2017). Therefore, changes in energy availability, both systemically and locally within cells and tissues, may influence the ability of the immune response to respond to vaccination. However, further study in this area is necessary.

In conclusion, there appeared to be little effect of vaccination with a multivalent respiratory vaccine on glucose, NEFA, and SUN concentrations in naïve beef steers. Rather, the effects of DEX administration, used to mimic acute and chronic stress, were much more pronounced, resulting in greater concentrations of glucose, NEFA, and SUN in DEX treated steers. These responses appear to mimic, in part, effects of acute and chronic glucocorticoid release in response to a stressor.