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. 2018 Dec 27;97(3):1171–1184. doi: 10.1093/jas/sky483

Effects of combined viral-bacterial challenge with or without supplementation of Saccharomyces cerevisiae boulardii strain CNCM I-1079 on immune upregulation and DMI in beef heifers

William Christian Kayser 1, Gordon E Carstens 1,✉, Kevin E Washburn 2, Thomas H Welsh Jr 1, Sara D Lawhon 3, Sanjay M Reddy 3, William E Pinchak 4, Eric Chevaux 5, Andrew L Skidmore 5
PMCID: PMC6396270  PMID: 30597005

Abstract

Objectives were to determine whether live yeast (LY) supplementation would affect daily dry matter feed intake, body weight (BW), immune, and febrile responses to a viral-bacterial (VB) respiratory challenge. Crossbred heifers (N = 38, BW = 230 ± 16.4 kg) were allocated into a 2 × 2 factorial treatment arrangement: Factor 1 = roughage-based diet with or without LY (Saccharomyces cerevisiae boulardii CNCM I-1079, 62.5 g/hd/d), Factor 2 = VB, intranasal administration of bovine herpesvirus-1 (BHV-1, 2 ×108, PFU) on day 0 and endobronchial inoculation with Mannheimia haemolytica (5.4 × 1010, CFU) on day 3, or intranasal saline administration followed by inoculation with phosphate buffer solution (PBS). Heifers were fed their respective diets for 27 d prior to VB challenge on day 0. Heifers were housed by treatment and group-fed using electronic feedbunks. Thermo-boluses (Medria; Châteaubourg, FR) measured rumen temperature (RUT) at 5-min intervals and rectal temperature and whole blood samples were collected on days 0, 3 to 8, 10, 13, and 15. Data were analyzed using repeated measures in the mixed procedure of SAS with fixed effects of day, diet, inoculation, and their interactions. Animals fed LY exhibited a 16% increase (P = 0.02) in neutrophils relative to CON. Diet × inoculation × day interactions were detected for monocytes and haptoglobin. The VB-LY had the greatest (P < 0.05) concentration of monocytes on day 4, followed by VB-CON which was greater (P < 0.05) than PBS treatments. Haptoglobin concentration was greatest (P < 0.02) for VB-CON on day 5, followed by VB-LY which was greater (P < 0.05) than PBS. Heifers supplemented with LY had less (P < 0.05) haptoglobin production than CON. The VB challenge produced nasal lesions that increased (P < 0.01) with day, reaching a zenith on day 6 with 70% of the nares covered with plaques, and increased (P < 0.05) neutrophils on days 3 to 5. The VB challenge increased RUT (P < 0.05) days 2 to 7 and rectal temperature (P < 0.05) on days 0 and 3 to 6. The increased rectal temperature on day 0 was likely due to increased ambient temperature at time of challenge, as VB heifers were processed after the PBS heifers to avoid contamination. The VB challenge was effective at stimulating immune responses, and RUT was effective for measuring febrile responses. These results indicate that prior LY supplementation altered the leukogram in response to VB challenge, suggestive of increased innate immune response.

Keywords: bovine herpes virus-1, bovine respiratory disease, Mannheimia haemolytica, rumen temperature, Saccharomyces cerevisiae boulardii

INTRODUCTION

Bovine respiratory disease (BRD) complex is one of the primary economic and animal welfare challenges facing beef producers (Rose-Dye et al., 2011; Griffin, 2014). Despite efforts to reduce mortality and morbidity through vaccination, antimicrobial therapy, and management strategies, mortality due to BRD in the feeding period is increasing and the leading cause of death for cattle in the United States (Engler et al., 2014; USDA 2015). The BRD complex is multifactorial and includes bacterial and viral pathogens that often coinfect animals, which results in increased severity of disease (Griffin et al., 2010).

Bovine herpes virus-1 (BHV-1) is a viral pathogen within the BRD complex that leads to upper respiratory tract disorders and exerts immunosuppressive effects, which increases susceptibility to secondary bacterial infection (Jones and Chowdhury 2007). Mannheimia haemolytica (MH) is the most prevalent bacterial pathogen associated with BRD (Smith 2015a). Although considered commensal, it is also opportunistic and is the most common isolate found in feedlot cattle with fatal fibrinous bronchopneumonia (Ackermann and Brogden 2000).

The US Food and Drug Administration’s (FDA) recent changes to the Veterinary Feed Directive Final Rule (2016) expressed interest in the use of antibiotic alternative solutions for treatment and prevention of BRD. This supports consumer concerns about the use of antibiotics in livestock production, which is evident by the rise in demand for natural and organic protein sources (O’Donovan and McCarthy 2002). In order for livestock producers to maintain a social license, while sustainably producing beef, antibiotic alternatives are a prerequisite (Brown and Nagaraja 2009). Direct-fed microbials (DFM) are potential antibiotic alternatives, and some research has demonstrated that they may be effective at reducing morbidity in shipped-stressed calves (Zinn et. al., 1999). Saccharomyces cerevisiae boulardii (live yeast [LY]) is one of the most widely investigated microorganisms due in part to its application to combat enteric diarrhea in humans; however, limited research has been conducted in ruminant animals (Łukaszewicz 2012).

Based on this need for antibiotic alternatives, the objectives of this experiment were 2-fold: first, to evaluate whether dietary supplementation with LY would influence immune response following the viral-bacterial (VB) challenge and potentially ameliorate the detrimental effects of the challenge; second, to develop a VB challenge model consisting of intranasal inoculation of BHV-1 followed by endoscopic inoculation of MH that would allow us to study immune and behavior changes associated with the acute response to BRD; and furthermore, to characterize deviations of the hemogram, circulating haptoglobin concentration and continuously measured physiological and behavior responses due to the VB challenge.

MATERIALS AND METHODS

All animal care and use procedures were in accordance with the guidelines for use of Animals in Agricultural Teaching and Research as approved by the Texas A&M University Institutional Animal Care and Use Committee (IACUC # 2015-0379) as well as the Texas A&M University Institutional Biosafety Committee (IBC # 2015-068).

Animals

Forty Angus crossbred heifers (initial body weight [BW] = 230 ± 16.4 kg) from the research cattle population at the Texas A&M University McGregor Research Center (McGregor, TX) were used in this study. The heifers were approximately 11 mo of age at the start of the study. At approximately 4 to 5 mo of age and again 3 wk prior to weaning, heifers were vaccinated for respiratory viral pathogens (Triangle 5; Boehringer Ingelheim Vetmedica, St. Joseph, MO; BHV-1, bovine viral diarrhea 1 and 2, bovine respiratory syncytial virus, and parainfluenza-3). The heifers were also vaccinated for Clostridial diseases (Covexin 8; Merck, Madison, NJ) at 4 to 5 mo of age, but were not previously vaccinated for MH prior to enrollment in the study.

All animals were considered to be clinically healthy based upon daily observations for 27 d prior to challenge. To qualify for the study, heifers had to be seronegative for MH and had to be seropositive to BHV-1. To accomplish this, serum samples were collected from 94 heifers to evaluate antibody titers against MH using the whole-cell agglutination test (Texas Veterinary Diagnostic Laboratory; TVMDL). The heifers selected for the study had MH antibody titers that were less than 11 dilutions, which is the positive threshold established by TVMDL (ranged from 7 to 10 dilutions; mean = 8.7). For BHV-1, antibody titers were assayed using a neutralization test, and those heifers with BHV-1 titers that ranged from 2 to 4 dilutions (mean = 2.7) were used in the study. Additionally, heifers were confirmed to be negative for persistently infected bovine viral diarrhea virus (BVDV) through analysis of ear notch samples that were analyzed using the BVD antigen-capture ELISA (BVD-Ag ELISA) method.

Experimental Design and Treatment Arrangements

Heifers were stratified by initial BW, MH and BHV-1 titer dilution, exit velocity, and prestudy average daily gain (ADG) and randomly assigned to 1 of 4 treatments arranged in a 2 × 2 factorial array. Factor 1 consisted of a roughage-based diet without (CON) or with added LY (Saccharomyces cerevisiae boulardii strain CNCM I-1079 at 62.5 g per heifer daily; Proternative Advantage; Lallemand Animal Nutrition, Milwaukee, WI). Factor 2 consisted of intranasal inoculation with BHV-1 on day 0 followed by bronchoselective endoscopic inoculation with MH (VB) on day 3 or intranasal inoculation with saline on day 0 followed by bronchoselective endoscopic inoculation with phosphate buffer solution (PBS) on day 3. Therefore, the 4 treatment arrangements were VB-CON, VB-LY, PBS-CON, and PBS-LY (n = 10).

The experiment period for the data presented in the current study was 22 d, which occurred within an 84-d period that was designed to evaluate treatment effects on growth and feed efficiency. Heifers were fed their respective diets for 27 d prior to BHV-1 challenge on day 0 and for 57 d after challenge. Throughout the study, all animals were housed in 1 of 8 pens equipped with electronic feedbunks (GrowSafe) at Texas A&M University’s McGregor Research Center in McGregor, TX, with 2 pen replicates per treatment. The 4 pens with the VB-CON and VB-LY treatments were separated by approximately 35 m from the 4 pens with the PBS-CON and PBS-LY pens to ensure that that VB- and CON-treated heifers did not have contact with each other or share water troughs. Furthermore, the VB-CON and VB-LY treatment pens were on the prevailing downwind side. Heifers were offered feed ab libitum once daily at 0700 h. The diet (DM basis) contained 36.5% dry rolled corn, 26% corn dried distillers’ grains, 30% chopped alfalfa hay, 5% molasses, and 2.5% dry mineral. Targeted intake of LY in this study was 62.5 g per heifer daily, which was premeasured and hand mixed in the diet prior to delivery. Similarly, 62.5 g per heifer of an isonitrogenous isocaloric placebo was included in the control diet daily. To prevent CON animals feed from being contaminated with LY each pen had dedicated feed bins where the LY or placebo was hand mixed prior to delivery. Feeders were blinded to dietary treatments, and the LY and CON diets were analyzed weekly for LY strain CNCM I-1079. The LY diet always contained colony forming units (CFU) at or above the prescribed level of CFU for this study of 2.5 × 1010 per day. Throughout the study, no CNCM I-1079 or wild yeast colonies were detected in the CON diet.

The inoculum for the VB challenge was prepared prior to inoculation (day 0). The Cooper strain of BHV-1 that was originally obtained from the United States Department of Agriculture’s (USDA) Center for Veterinary Biologics (CVB) in Ames, IA was used in this study. Briefly, the virus was propagated by inoculating 12 roller bottles that had a surface area of 850 cm2 with Madin Darby bovine kidney (MDBK) cells. Cells were grown in Eagles minimum essential medium (EMEM) with Earles basic salt and 10% fetal bovine serum (FBS). Growth medium was poured off and the BHV-1 inoculated at 10–1 dilution onto roller bottles and allowed to adsorb for 1 h at 36 ± 2 °C in 5 ± 1% CO2. After which, EMEM with Earles salts and 2% FBS were added and roller bottles were returned to 36 ± 2 °C in 5 ± 1% CO2 until cytopathic effect was observed to be approximately 90%. Virus was harvested by freeze/thaw, and the fluid containing the virus was centrifuged at low speed (500 × g for 20 min) harvested and stored at −70 °C. Virus was distended in media to achieve the desired concentration of 1 × 108 plaque forming units (PFU) per mL.

The MH inoculum was prepared as described by Mosier et al. (1995). Briefly, MH serotype A1 was grown on trypticase soy agar containing 5% sheep blood for 18 h at 37 °C in 5% CO2. Colonies were inoculated into brain-heart infusion broth and incubated for 16 to 18 h at 37 °C with aeration. The bacteria were then centrifuged at 3,000 × g for 15 min at 4 °C and washed with PBS twice. After the second wash, the bacteria were centrifuged as before, and the pellet was resuspended in PBS at a final concentration of 5.4 × 1010 CFU/10-mL dose. After preparation, the inoculum was placed on ice in a dark cooler and transported to the site of inoculation (approximately 174 km). After the inoculation was performed, a sample of the MH inoculum was returned to the pathobiology laboratory to ensure that the concentration of MH was consistent.

On day 0 prior to feed delivery, 1 mL of either BHV-1 at 1 × 108 PFU/mL or saline was aerosolized into each naris with a 3-mL syringe fitted with and intranasal mucosal atomization device (MAD Nasal; Teleflex, Morrisville, NC). Heifers in the PBS treatment were inoculated prior to heifers in the VB treatment and the chute and processing area were disinfected after BHV-1 inoculation. On day 3 prior to feed delivery, all heifers were brought back to the process facility and endoscopically inoculated with MH or PBS. To avoid any chance of PBS animals receiving MH via contamination of the instruments used for inoculation, the PBS treatment group was inoculated prior to the MH treatment group. The inoculations were performed with an endoscope as described by Theurer et al. (2013). Heifers were captured in a standard squeeze chute and their heads were restrained with a halter specifically designed for cattle. An endoscope 1 m in length was inserted into the ventral meatus of one nostril and passed into the trachea to the level of the right apical lung lobe bronchi allowing visualization of the opening. A sterile bronchoalveolar lavage tube was inserted into the endoscope portal and passed until the tip of the lavage tube was visible emerging from the endoscope. The lavage tube was advanced another 1 to 2 cm into the opening of the right apical lung lobe bronchi. Once the lavage tube was in place, heifers in the PBS treatment group were administered 10 mL of PBS followed by a 60-mL flush of PBS for a total of 70 mL. Following inoculation of PBS animals, the endoscope was disinfected with chlorhexidine solution and rinsed with saline. Subsequently, heifers in the MH treatment groups were challenged with 10 mL of M. haemolytica serotype A1 at 5.4 × 1010 CFU/mL followed by 60 mL of PBS for a total of 70 mL. No adverse effects due to the inoculation procedure were observed for either treatment.

Data Collection

Temperature monitoring, nasal lesion, and clinical illness scoring

Rectal temperature was recorded using a digital thermometer (Cooper TM99A, Cooper-Atkins Corporation, Middlefield, CT) on days −6, 0, 3, 4, 5, 6, 8, 10, 13, and 15. In addition, radiofrequency biothermal boluses (ThermoBolus, Medria, Châteaubourg, France) were inserted into the rumen of all heifers to continuously recorded reticulo-rumen temperature (RUT) at 5-min intervals. A proprietary algorithm was used to remove variation in RUT due to drinking events. Summary statistics of RUT were computed on a daily basis for the duration of the experiment. After inoculation with BHV-1, the mucosa of the nares was observed daily to quantify the formation of BHV-1 plaques. Nasal lesions were subjectively evaluated by 1 observer based on the percentage of visible naris covered with plaques in 10% increments until all nasal plaques were resolved (day 13). Heifers were monitored by 2 experienced evaluators twice daily for the duration of the experiment for clinical signs consistent with BRD. The visual evaluation employed in the experiment has been described in detail by Step et al. (2008). The criteria include signs of depression, inappetence, and respiratory distress. The evaluators assigned a severity score of 1 to 4, where 1 was assigned for mild, 2 for moderate, 3 for severe, and 4 for moribund. Heifers receiving a 3 or greater were pulled from the pen and given a full medical evaluation. Rectal temperature was measured during the medical evaluation and if it exceeded 40.5 °C antimicrobial therapy was administered. All heifers were returned to their home pen after the evaluation. Temperature readings, BW, and treatments were recorded for every animal that was examined for clinical signs consistent with BRD. The first treatment administered to heifers suffering from BRD was tulathromycin (Draxxin, Zoetis, Parsippany, NJ) at 2.5 mg/kg BW. If the initial treatment was ineffective after 7 d, ceftiofur hydrochloride (Excenel, Zoetis, Parsippany, NJ) was administered at 2.2 mg/kg BW.

Serum haptoglobin and cortisol

Heifers were restrained in a squeeze chute and their heads were restrained with a halter to collect blood samples (10 mL; Vacutainer with no additive, Becton, Dickson and Company, Franklin Lakes, NJ) via jugular venipuncture (18-gauge needle) on days −6, 0, 3, 4, 5, 6, 8, 10, 13, and 15, relative to BHV-1 challenge. After collection, all samples were immediately placed on ice. All day 0 samples were collected prior to BHV-1 challenge and all day 3 samples were collected prior to MH inoculation. To harvest serum, samples were centrifuged at 3,000 × g for 20 min at 20 °C and stored in duplicate aliquots at −20 °C until subsequent analysis. Haptoglobin was measured with a commercial, bovine-specific sandwich ELISA kit (Immunology Consultants Laboratory, Inc., Portland, OR). Furthermore, the concentration was determined by the average of duplicate unknown samples compared with a standard curve (4-parameter logistic) generated from known concentrations of bovine haptoglobin using Assay Zap software (Biosoft, Cambridge, UK). The haptoglobin analysis had an interassay CV of 3.81%. Serum concentrations of cortisol were determined as described by Littlejohn et al. (2016). A solid phase radioimmunoassay (DSL-2100; Diagnostic Systems Labs, Webster, TX) using antiserum-coated tubes were prepared according to the manufacturer’s directions. Serum cortisol concentrations were determined by the average of duplicate unknown samples compared with a standard curve generated from known concentrations of cortisol using Assay Zap software (Biosoft, Cambridge, UK). The minimum detectable cortisol concentration for this assay was 1.02 ng/mL, and the interassay CV was 11.4%.

Hemogram

Blood samples (7 mL; EDTA, Becton, Dickson and Company, Franklin Lakes, NJ) were collected via jugular venipuncture with an 18-gauge needle on days −6, 0, 3, 4, 5, 6, 8, 10, 13, and 15, relative to BHV-1 challenge. Samples were immediately submitted to a commercial lab (Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, TX) for total leukocyte, erythrocyte, hematocrit, hemoglobin, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and platelets. Blood counts were performed with an automated hemocytometer (ADVIA 120, Siemens Healthcare Diagnostics, Tarrytown, NY) using the factory-installed cattle setting (ADVIA 120 Multispecies System Software, Version 2.206 MS, Siemens Healthcare Diagnostics). The hemocytometer counts leukocytes, erythrocytes, and platelets by optical scatter and fluorescence. Hemoglobin concentration is determined by the cyano-methemoglobin technique. Differential leukocyte percentages were determined by counting cells on modified blood smears and absolute counts were calculated using the total leukocyte count from the hemocytometer.

Feed intake

All pens were equipped with electronic feedbunks (GrowSafe Systems Ltd., Airdrie, AB, Canada) to facilitate collection of feed intake on an individual-animal basis. The GrowSafe system consisted of feed bunks equipped with load bars to measure feed disappearance, and an antenna located within each feed bunk to record animal presence via detection of EID tags. Assigned feed disappearance (AFD), which is a metric to evaluate the quality of the feed intake data (Proportion of feed delivered that was assigned to animals), was computed daily for each feed bunk, and data for the pen deleted from the analysis if the AFD was <95% for the pen, or if the system malfunctioned. For the study, feed intake data were deleted for 4 d from Pen 1 (days −3, −2, 4, and 8), 5 d from Pen 3 (days 2, 4, 10, 11, and 12), 3 d from Pen 5 (days 11, 12, and 13), and 2 d from Pen 8 (days 12 and 13). However, feed intake data were available for each day of the study from at least 1 of the 2 replicate pens per treatment. A subroutine of the GrowSafe 6000E software (Process Feed Intakes) was used to compute individual-animal feed intake based on continuous recordings of feed disappearance during each bunk visit event.

Statistical Analysis

This experiment was designed as a randomized complete block with a 2 × 2 factorial treatment arrangement with animal serving as the experimental unit. Growth rates for individual heifers were modeled by linear regression of BW measurements on study day using PROC GLM (SAS, 9.4). The regression coefficients were used to compute ADG, initial BW, and final BW. Temperature, feed intake, haptoglobin, cortisol, and hemogram data were analyzed using the MIXED procedure (SAS 9.4, SAS Institute Inc., Cary, NC) with an autoregressive covariance structure. The model for all variables included the main effects of diet, inoculation, day, and all possible interactions. Although heifers were randomized into treatments with prestudy ADG (days −58 through −31) as a consideration, there were differences in ADG between treatments measured from days −27 through 0 of the study. Therefore, initial ADG measured during the 27 d prior to challenge was used as a covariate to remove any effects of ADG on dependent variables. When inoculation treatment × day or dietary treatment × day interactions were detected (P ≤ 0.05), the SLICE output option was used to identify treatment differences within day. In the event that diet × inoculation or diet × inoculation × day interactions were detected (P ≤ 0.05), treatment least squares means were evaluated using the PDIFF multiple comparison test.

RESULTS

Of the 40 heifers initially enrolled in the study, 2 heifers (1 VB-LY and 1 PBS-LY) had to be removed due to lameness. Initial (day −6) and final (day 15) BW were not affected by the inoculation or dietary treatments, and there were no interactions detected for BW. The dietary treatment did not affect ADG, and there was no diet × inoculation treatment interaction detected for ADG. However, as expected, VB-challenged heifers exhibited reduced (P < 0.01) ADG of 0.31 kg/d compared with 1.21 kg/d for the PBS heifers. Furthermore, an inoculation × day interaction (P < 0.01) was detected for BW (Figure 1); however, mean separation between inoculation treatments was not significant within day. Caution should be used when interpreting these results because ADG and BW were measured over a relatively short 22-d time period. Dietary treatment did not affect dry matter feed intake (DMI) and the dietary treatment × day interaction was not detected for DMI. However, there was an inoculation treatment × day interaction (P < 0.01) for DMI, with VB heifers having reduced DMI on days 3, 4, 5, 6, and 7 (Figure 1) compared with PBS-inoculated heifers.

Figure 1.

Figure 1.

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Model-adjusted least squares mean estimates for daily dry matter feed intake (DMI) (above) and body weight (below) by day relative to experimental inoculation with viral-bacterial (VB; bovine herpes virus-1 [BHV-1] on day 0 followed by Mannheimia haemolytica on day 3) challenge or phosphate buffer solution (PBS; negative control).The model included effects for study day and repeated measures on individual heifers with pre-ADG as a covariate, and the SLICE option was utilized in the MIXED procedure to determine significance within day. ♦Significant differences (P < 0.05) between treatments groups within study day. The inoculation treatment × day interaction was significant (P < 0.001) for both DMI and body weight.

There were no differences detected in clinical illness scores due to inoculation treatment. In fact, only 2 heifers (VB treatment) were assessed a clinical illness score ≥ 3. However, the rectal temperatures of these heifers did not exceed 40.5 °C; therefore, drug therapy was not administered. Furthermore, there were no naturally occurring cases of BRD, or repulls or mortalities due to the VB challenge during the study. Prior to BHV-1 administration, the mucosal surface of the nares of all heifers was inspected and found to be free of lesions. During the study, no nasal lesions were observed in the PBS-treatment heifers, and no difference in the prevalence of nasal lesions was detected due to dietary treatment. However, there was a significant (Figure 2; P < 0.01) inoculation treatment × day interaction for nasal lesions. Heifers inoculated with BHV-1 had greater (P < 0.01) prevalence of nasal lesions from days 3 to 11, reaching a zenith on day 6 post-BHV-1 challenge, with approximately 70% of the visible nares covered in plaques. Figure 3 is a photographic presentation of the formation of nasal lesions for 1 of the VB-treatment heifers, which was representative of the nasal lesions observed during the VB challenge.

Figure 2.

Figure 2.

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Model-adjusted least squares mean estimates for nasal lesions (top), rectal temperature (middle), and rumen temperature (RUT; bottom) by day relative to experimental inoculation with viral-bacterial (VB; bovine herpes virus-1 [BHV-1] on day 0 followed by Mannheimia haemolytica on day 3) challenge or phosphate buffer solution (PBS; negative control). Model included effects for study day and repeated measures on individual heifers with pre-ADG as a covariate, and the SLICE option was utilized in the MIXED procedure to determine significance within day. ♦Significant differences (P < 0.05) between treatments groups within study day. The inoculation × day interaction was significant for all dependent variables (P < 0.001).

Figure 3.

Figure 3.

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Progression of bovine herpes virus-1 plaques on the mucosa of the left naris, from day 3 through day 10 following intranasal inoculation.

Dietary treatment did not affect rectal temperature or RUT, and there were no diet × day interactions detected for either rectal temperature or RUT (Figure 2). There was an inoculation treatment × day interaction (P < 0.01) detected for rectal temperature, with VB-challenged heifers having greater rectal temperature on days 0, 3, 4, 5, and 6. Similarly, there was an inoculation treatment × day interaction (P < 0.01) detected for RUT, with VB-challenged heifers exhibiting greater RUT on days 2, 3, 4, 5, 6, and 7, which reached a zenith on day 4 with an average RUT of 41.0 °C. In addition, the VB-challenged heifers exhibited a decreased RUT on days 9, 10, 11, and 12 compared with the PBS-treated heifers.

Hemogram least square means for the dietary and inoculation treatments as well as P values for all possible interactions are reported in Table 1. Heifers supplemented with LY had a 16.4% greater (P < 0.02) concentration of neutrophils compared with the CON heifers. However, there were no other main effect differences in hemogram constituents due to LY supplementation. There was a diet × inoculation × day interaction (P < 0.01) detected for monocyte concentration (Figure 4; Table 2). On the day following MH challenge (day 4), heifers in the VB-LY treatment had the greatest (P < 0.05) concentration of circulating monocytes at 1.93 k cells/μL. On days 4, 5, 6, and 8, the VB-CON and VB-LY heifers had greater (P < 0.05) monocyte concentration than the PBS-CON and PBS-LY heifers, although the PBS-LY heifers did not differ from the VB-challenged heifers on days 5 and 8. On day 4, the VB challenge-induced increase in monocyte concentration was greater (P < 0.05) in VB-LY than VB-CON heifers, with heifers in both VB treatments having greater (P < 0.05) monocyte concentration than PBS heifers.

Table 1.

Main effects of dietary treatment and inoculation on hemogram constituents, haptoglobin, and cortisol concentrations

Item Diet1 Inoculation2 Interaction3P values
CON LY SE P VB PBS SE P In*Diet In*D Diet*D In*Diet*D
N 20 18 – – 19 19 – – – – – –
Eosinophil, K/µL 0.33 0.32 0.04 0.83 0.28 0.37 0.05 0.09 0.21 0.09 0.68 0.24
Lymphocytes, K/µL 5.03 4.88 0.19 0.44 4.85 5.06 0.19 0.27 0.25 0.01 1.00 0.81
Neutrophils, K/µL 4.40 5.12 0.29 0.02 5.56 3.96 0.29 0.01 0.08 0.01 0.46 0.59
Monocytes, K/µL 0.70 0.75 0.05 0.29 0.83 0.62 0.05 0.01 0.58 0.01 0.72 0.01
Leukocytes, K/µL 10.5 11.1 0.47 0.16 11.5 10.1 0.47 0.05 0.60 0.01 0.43 0.28
Hemoglobin, g/dL 12.0 12.1 0.19 0.72 12.1 11.9 0.19 0.33 0.99 0.01 0.33 0.16
Platelets, K/µL 637 627 33.5 0.77 634 630 33.5 0.91 0.89 0.01 0.96 0.94
Erythrocytes, M/µL 7.54 7.46 0.17 0.59 7.50 7.50 0.17 0.95 0.42 0.01 0.84 0.09
Hematocrit, % 0.33 0.33 0.01 0.90 0.33 0.32 0.01 0.27 1.00 0.01 0.57 0.06
MCH4, pg 15.9 16.2 0.24 0.15 16.2 15.9 0.24 0.17 0.09 0.68 0.76 0.78
MCV5, fL 43.3 43.9 0.86 0.44 44.1 43.2 0.86 0.31 0.24 0.10 0.91 0.61
MCHC6, g/dL 36.8 37.0 0.12 0.16 36.9 36.9 0.12 0.53 0.88 0.03 0.61 0.48
Haptoglobin, mg/dL 5.67 3.91 1.74 0.32 9.56 0.02 1.74 0.01 0.31 0.01 0.02 0.02
Cortisol, ng/mL 24.0 27.2 3.23 0.33 25.8 25.3 3.94 0.90 0.24 0.29 0.79 0.70
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The P-value threshold for significance is 0.05.

1Diet: CON = control (negative control); LY = supplementation with Saccharomyces cerevisiae boulardii at 62.5 g/hd daily.

2Inoculation: VB= viral-bacterial challenge which consisted of inoculation with bovine herpes virus-1 followed by Mannheimia haemolytica on day 3; PBS = phosphate buffer solution (negative control).

3Interactions: In*Diet = Inoculation × Diet; In*D = Inoculation × Day; Diet*D = Diet × Day; In*Diet*D = Inoculation × Diet × Day.

4MCH = mean corpuscular hemoglobin.

5MCV = mean cell volume.

6MCHC = mean corpuscular hemoglobin concentration.

Figure 4.

Figure 4.

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Model-adjusted least squares mean estimates of monocytes for the interaction of inoculation × diet × day. With day relative to experimental inoculation with viral-bacterial (VB; bovine herpes virus-1 [BHV-1] on day 0 followed by Mannheimia haemolytica on day 3) challenge or phosphate buffer solution (PBS; negative control). The model included effects for study day and repeated measures on individual heifers with pre-ADG as a covariate. The inoculation × diet × day interaction was significant (P < 0.001). CON = control diet and LY = supplementation with Saccharomyces cerevisiae boulardii.

Table 2.

Least square mean estimates for monocytes1 following experimental viral-bacterial challenge

Day relative to VB challenge
Treatment2 −6 0 3 4 5 6 8 10 13 15
VB-CON 0.39 0.81 0.72 1.35b 0.89b 1.10b 0.71ab 0.85 0.71 0.55
VB-LY 0.51 0.86 0.98 1.93c 0.88b 1.29b 0.82b 0.68 0.54 0.62
PBS-CON 0.48 0.51 0.72 0.82a 0.51a 0.72a 0.43a 0.77 0.66 0.53
PBS-LY 0.46 0.54 1.00 0.50a 0.73ab 0.62a 0.69ab 0.78 0.57 0.37
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a–cWithin column, rows with different subscripts differ (P < 0.05).

1Presented means are in K cells/μL.

2Treatment: VB-CON = viral-bacterial challenge which consisted of inoculation with bovine herpes virus-1 followed by Mannheimia haemolytica on day 3 and control diet (negative control); VB-LY = viral-bacterial challenge and supplementation with Saccharomyces cerevisiae boulardii a 62.5 g/hd daily (LY); PBS-CON = phosphate buffer challenge (negative control) and control diet; PBS-LY = phosphate buffer challenge and supplementation with LY.

Interaction of diet × inoculation × day was significant at P < 0.01

Similar to monocytes, a diet × inoculation × day interaction (P < 0.02) was detected for serum haptoglobin concentration (Figure 5; Table 3). Following inoculation, VB-challenged heifers exhibited increased (P < 0.01) serum concentrations of haptoglobin, whereas serum concentrations of haptoglobin in PBS-treated heifers never exceeded 0.15 mg/dL. On day 5, heifers in the VB-CON treatment had the greatest (P < 0.05) serum haptoglobin concentration of 50.2 mg/dL (Table 3), which was greater (P < 0.05) than the VB-LY treatment (15.1 mg/dL). Both VB treatments were greater (P < 0.05) than the PBS treatments on day 5, whose serum haptoglobin concentrations were ≤ 0.01 mg/dL. On day 6, VB-LY and VB-CON had serum haptoglobin concentrations of 35.0 and 25.1 mg/dL, respectively, which were greater (P < 0.05) than both PBS treatments. The serum haptoglobin concentrations did not differ between VB-challenged and PBS-treated heifers on days 8 and 10.

Figure 5.

Figure 5.

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Model-adjusted least squares mean estimates of haptoglobin for the interaction of inoculation × diet × day. With day relative to experimental inoculation with viral-bacterial (VB; bovine herpes virus-1 (BHV-1) on day 0 followed by Mannheimia haemolytica on day 3) challenge or phosphate buffer solution (PBS; negative control). The model included effects for study day and repeated measures on individual heifers with pre-ADG as a covariate. The inoculation × diet × day interaction was significant (P < 0.02). CON = control diet and LY = supplementation with Saccharomyces cerevisiae boulardii.

Table 3.

Least square mean estimates for haptoglobin1 following experimental viral-bacterial challenge

Day −6 0 3 4 5 6 8 10
VB-CON 1.61 0.03 0.00 4.74 50.19c 25.12b 8.27 0.74
VB-LY 0.79 0.02 0.01 2.39 15.09b 34.95b 7.83 1.19
PBS-CON 0.00 0.00 0.00 0.00 0.00a 0.01a 0.01 0.00
PBS-LY 0.02 0.00 0.00 0.00 0.01a 0.00a 0.03 0.15
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a–cWithin column, rows with different subscripts differ (P < 0.05).

1Presented means are in mg/dL.

2Treatment: VB-CON = viral-bacterial challenge which consisted of inoculation with bovine herpes virus-1 followed by Mannheimia haemolytica on day 3 and control diet (negative control); VB-LY = viral-bacterial challenge and supplementation with Saccharomyces cerevisiae boulardii a 62.5 g/hd daily (LY); PBS-CON = phosphate buffer challenge (negative control) and control diet; PBS-LY = phosphate buffer challenge and supplementation with LY.

Interaction of diet × inoculation × day was significant at P < 0.01.

Leukogram constituents that were significantly (P < 0.05) affected by inoculation treatment × day interactions are presented in Figure 6. Leukocyte count was greater (P < 0.05) for VB-challenged heifers on days 4, 5, and 15, compared with the PBS-treated heifers. The VB challenge reduced (P < 0.05) lymphocytes on days 4, 8, 10, and 15 relative to the PBS-treated heifers. Similar to leukocytes, neutrophils were increased in VB-challenged heifers from days 3 to 5, reaching a zenith on day 4. Neutrophils were also increased (P < 0.05) in the VB-challenged heifers on days 10, 13, and 15 compared with the PBS-treated heifers, although the magnitude of difference was less.

Figure 6.

Figure 6.

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Model-adjusted least squares mean estimates for leukocytes (top), lymphocytes (middle), and neutrophils (bottom) by day relative to experimental inoculation with viral-bacterial (VB; bovine herpes virus-1 [BHV-1] on day 0 followed by Mannheimia haemolytica on day 3) challenge or phosphate buffer solution (PBS; negative control). Model included effects for study day and repeated measures on individual heifers with pre-ADG as a covariate, the SLICE option was utilized in the MIXED procedure to determine significance within day. ♦Significant differences (P < 0.05) between treatments groups within study day. The inoculation × day interaction was significant for all dependent variables (P < 0.001).

Alterations to the erythron with significant (P < 0.01) inoculation treatment × day interactions are presented in Figure 7. Even though the day × inoculation interaction for platelets was significant (P < 0.01), the results are not presented, due to there being no differences between the treatments on a given day. Total erythrocyte count was reduced (P < 0.05) on days 8, 10, 13, and 15, for VB-challenged heifers compared with the PBS heifers. Hemoglobin was increased (P < 0.05) for VB-challenged heifers on day 4 and decreased (P < 0.05) on days 10 and 13, compared with the PBS heifers. Heifers in the VB challenge exhibited reduced hematocrit on days 6, 8, 10, 13, and 15, and increased MCHC on days 4, 5, 6, and 15 compared with PBS heifers.

Figure 7.

Figure 7.

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Model-adjusted least squares mean estimates for erythrocytes (A), hemoglobin (B), hematocrit (C), and mean corpuscular hemoglobin concentration (MCHC; D) by day relative to experimental inoculation with viral-bacterial (VB; bovine herpes virus-1 [BHV-1] on day 0 followed by Mannheimia haemolytica on day 3) challenge or phosphate buffer solution (PBS; negative control). The model included effects for study day and repeated measures on individual heifers with pre-ADG as a covariate, and the SLICE option was utilized in the MIXED procedure to determine significance within day. ♦Significant differences (P < 0.05) between treatments groups within study day. The inoculation × day interaction was significant for all dependent variables (P < 0.001).

DISCUSSION

Although previous reports have utilized a similar (BHV-1 + MH) combined VB challenge model, typically the MH is delivered intratracheally or aerosolized (Jericho and Langford, 1978; Stabel et al., 1993; Word et al., 2016a). In the current study, MH was delivered endobronchially and the only other example in the literature was performed on Holstein calves of ≤5 mo of age (Narita et al., 2000). Therefore, the objectives of this study were to evaluate the challenge model on animals that would be typical of feedyard placements in the United States, and to evaluate whether prior supplementation of LY would ameliorate the negative effects of the VB challenge on physiological, immune, and behavioral responses.

Common clinical signs associated with naturally occurring BRD include pyrexia, lethargy, anorexia, and dyspnea (Duff and Galyean 2007). The current challenge model did not create gross signs of clinical disease, which was not unexpected as previous studies using a similar MH strain with intratracheal delivery have reported that challenged animals appeared clinically normal or displayed mild increases in clinical illness scores that were not different from clinically normal (Corrigan et al., 2007; Capik et al., 2015). However, VB-challenged heifers had marked reductions in DMI following MH inoculation. Reduced feed intake has been well documented as an early biomarker of BRD in studies with high-risk calves (Hutcheson and Cole, 1986; Galyean and Hubbert, 1995; Sowell et al., 1999; Quimby et al., 2001). Theurer et al. (2013) reported reductions in the time spent at a hay feeder in calves that were similarly inoculated with MH. Although fluctuations in bunk visit duration are not equivalent to changes in DMI, these 2 traits are moderately correlated (0.37 to 0.52; Kayser and Hill 2013) such that reductions in duration of feeding were likely associated with decreased feed intake. In the current study, inoculation with BHV-1 did not affect DMI on days 0 to 2, which agrees with Reffett et al. (1988) and Stabel et al. (1993). Both studies administered a BHV-1 challenge to Holstein calves and reported no differences in feed intake following inoculation. Similarly, Burciaga-Robles et al. (2010a) reported no differences in DMI between steers exposed to a BVD persistently infected steer and negative controls. In contrast to these reports, Cole et al. (1992) reported decreases in daily feed intake in steers that were challenged with BHV-1. However, the impact of the challenge did not appear to reduce feed intake until 3 d after administration. The impact that BHV-1 challenge exerts on DMI is unclear; in the current study, we are only able to evaluate the time between the inoculations in which there was no difference. The reduced DMI resulting from the VB challenge was synonymous with naturally occurring BRD cases, which illustrates the effectiveness of the model.

Clinical symptoms of the respiratory form of BHV-1 share commonality with other pathogens in the BRD complex such as hyperthermia, anorexia, coughing, nasal discharge, and excess salivation (Jones and Chowdury 2007). However, unique to BHV-1 is the formation of plaques on the mucosa of the nares and trachea (Yates 1982). Furthermore, McKercher et al. (1957) reported that naturally occurring and experimentally induced BHV-1 lesions are similar; however, experimentally induced BHV-1 cases had increased severity of rhinitis and decreased tracheitis relative to naturally occurring cases (Yates, 1982). In the current study, BHV-1 inoculation created characteristic plaques and erosions on the mucosal surface of the nares, as well as localized inflammation of the nares (Gershwin et al., 2015). Similarly, Word et al. (2016b) reported nasal lesion formation following inoculation with BHV-1, and previous supplementation with live yeast and yeast cell wall tended to decrease nasal lesions scores (2.5 vs. 3.2, respectively). In the current study, supplementation with LY did not affect lesion formation.

Rectal temperature is typically used as an objective method to diagnose BRD cases. The VB challenge induced hyperthermia as measured by both rectal temperature and RUT. Increases in core body temperature due to the BHV-1 challenge were observed beginning on day 2 for RUT and on day 3 for rectal temperature. It is possible that rectal temperature may have increased prior to day 3; however, daily rectal temperatures were not measured in this study to minimize stress. A similar febrile response was reported by Cole et al. (1992), whereby rectal temperature increased as a result of BHV-1 inoculation beginning on day 3, although rectal temperature only exceeded 40 °C for 24 h. Rose-Dye et al. (2011) reported no change in rectal temperature or RUT due to a 72-h exposure with a BVD persistently infected calf; however, inoculation with MH stimulated an almost immediate increase in both rectal temperature and RUT. Numerous studies have reported an increase in core body temperature due to an experimental MH challenge, with durations of elevated body temperature occurring for 24 to 36 h (Corrigan et al., 2007; Burciaga-Robles et al., 2010b; Hanzlicek et al., 2010; Theurer et al., 2013; Capik et al., 2015). In the current study, the increase in core body temperature was observed for longer durations compared with these single-pathogen challenge studies. The VB-challenged heifers had greater RUT for 6 d and greater rectal temperature for 4 d relative to the PBS-treated heifers. The greater magnitude and duration of the hyperthermic response observed in this study compared with previous studies suggest that the combined-VB challenge induced an additive febrile response. Interestingly, the VB-challenged heifers actually had lower RUT during days 9 to12 than PBS-treated heifers. Although the causation for this apparent hypothermic response in VB-challenged heifers remains unclear, it may represent a biphasic thermoregulatory response to cope with an energy imbalance created by the reduction in DMI and the hyperthermic response induced by the VB challenge (Romanovsky et al., 1996).

The increased rectal temperature observed in VB-challenged heifers on the day 0 may have been related to increased ambient temperature at the time of inoculation, as VB-challenged heifers were processed after the PBS-treated heifers to reduce the potential of contamination. Interestingly, there was no difference in RUT between the treatments on day 0, which likely reflects the fact that RUT was continuously measured at 5-min intervals, with daily estimates based on the average of 288 measurements, whereas rectal temperature was based on a single-point measurement. Hanzlicek et al. (2010) measured rectal temperature 3 times daily in beef steers that were experimentally challenged with MH and found that rectal temperature was greater in the early evening (40.2 °C) compared with measurements obtained in the morning (39.6 °C) or at noon (39.4 °C). These results reinforce that the VB challenge effectively stimulated a physiological response and suggest that RUT may be a robust tool for identifying febrile animals.

The VB-challenge heifers had increased leukocyte concentrations that would be classically associated with upregulation of the innate immune system due to an acute infection. Interestingly, the leukogram appears to have only been affected by the MH inoculation. With the exception of neutrophils, there were no differences in any of the leukocyte or erythrocyte concentrations on day 3. However, the day following MH inoculation (day 4), almost all of the variables were affected by the VB-challenge treatment. Monocytes were increased the day following MH inoculation and were greater for the heifers fed LY. The impact of experimental challenge on monocytes is inconclusive. Similar to the current study, Corrigan et al. (2007) reported an increase in monocytes following inoculation with MH. However, Hanzlicek et al. (2010) reported no difference in monocytes resulting from MH challenge. Furthermore, Burciaga-Robles et al. (2010b) and Gånheim et al. (2005) reported no differences in calves challenged with BVD, MH, or a combination of both. Monocytes are immature-circulating macrophages which are transported to tissues during infection or inflammation, upon entering a tissue monocytes differentiate into macrophages and are the major phagocyte population resident in normal tissues at homeostasis (Murphy and Weaver 2017). The increase in monocytes following challenge suggests that the model effectively stimulated an upregulation of the innate immune system. The increased monocytes observed in heifers fed LY also suggest that LY increased the innate immune response, which would allow for increased phagocytic activity within the infected area.

The acute phase response is induced by proinflammatory cytokines, which are protein hormones that act as messengers between local site of injury and hepatocytes which synthesize the acute phase proteins (Petersen et al., 2004). Haptoglobin is a positive acute phase protein that binds free hemoglobin in blood circulation and creates a haptoglobin–hemoglobin complex, which is thought to sequester and limit the amount of Fe available for bacterial proliferation (Petersen et al., 2004; Richeson et al., 2016). Heifers in the VB challenge exhibited a rapid increase in serum haptoglobin concentration following the MH inoculation. The increase in haptoglobin due to MH inoculation is often reported and repeatable (Corrigan et al., 2007; Burciaga-Robles et al., 2010b; Theurer et al., 2013). This increase in serum haptoglobin concentration suggests that the animal’s immune system stimulated an acute phase protein response, and validates the challenge model. Interestingly, in the current study, LY supplementation delayed the zenith of the haptoglobin by 1 d. Furthermore, heifers supplemented with LY had overall less haptoglobin produced than the controls. Similarly, Word et al. (2016b) reported that supplementation with a combined live Saccharomyces cerevisiae and cell wall extract tended to reduce serum haptoglobin concentration in heifers challenged with BHV-1 and MH. Results from these studies suggest that the LY supplementation may reduce the inflammatory response, and the subsequent catabolic effects associated with the acute phase response.

Total leukocytes were increased the day following inoculation with MH, which agrees with previous studies (Gånheim et al., 2005; Corrigan et al., 2007; Hanzlicek et al., 2010; Burciaga-Robles et al., 2010b). In the current study, slight neutrophil increase due to BHV-1 inoculation was detected on day 3. Conversely, Gånheim et al. (2005) reported no changes in circulating neutrophils due to BVD challenge, and Burciaga-Robles et al. (2010b) reported a reduction in neutrophils due to BVD exposure. The impact that viral challenges have on neutrophil production is unclear. However, neutrophilia in response to MH challenge is repeatable and indicates challenge success (Gånheim et al., 2005; Corrigan et al., 2007; Hanzlicek et al., 2010; Burciaga-Robles et al., 2010b). Heifers supplemented with LY had greater overall circulating neutrophils compared with the controls. Contrary to these findings, Word et al. (2016b) reported that supplementation with Saccharomyces cerevisiae tended to reduce neutrophils relative to controls in a combined BHV-1 + MH challenge. Differences between these results could be situational, or due to variation between the strains of live yeast. In the current study, the subspecies boulardii was supplemented, which is genetically indistinguishable but has different growth patterns than cerevisiae (Fietto et al., 2004). Although there was no effect of day, supplementation with LY may improve the efficacy of the innate immune response through increased neutrophil concentration.

Transient lymphopenia in ruminants is commonly induced during the acute phase of systemic infectious diseases (Smith 2015b). In the current study, the VB challenge decreased circulating lymphocyte concentrations. Similarly, Burciaga-Robles et al. (2010b) and Gånheim et al. (2005) reported decreases in lymphocytes due to MH and BVD challenges. However, Corrigan et al. (2007) and Hanzlicek et al. (2010) reported no changes in circulating lymphocytes following MH challenge. The cause of the inconsistency between the studies is unknown; however, the reductions in lymphocytes in the current study further validate the challenge model’s ability to illicit an immune response.

The reductions in erythrocytes and hemoglobin from the VB challenge are most likely associated with inflammation of lung tissue. The mild anemia resulting from MH challenge has been reported previously (Corrigan et al., 2007; Hanzlicek et al., 2010; Burciaga-Robles et al., 2010b). Hematocrit is calculated as the proportion of erythrocytes to MCV. There were no differences in MCV throughout the study, and the differences in hematocrit are likely due to reductions in erythrocytes. Similarly, MCHC is computed as the proportion of hemoglobin to hematocrit and the observed differences are a result of decreased erythrocyte and hemoglobin concentrations.

CONCLUSIONS

The VB challenge model successfully stimulated an innate immune response, which resulted in rhinitis with lesions, hyperthermia, increases in circulating haptoglobin, and alterations to the hemogram synonymous with an acute respiratory tract infection. Furthermore, the VB challenge reduced DMI and suppressed ADG. Results from this study highlight the value of continuous remote monitoring of RUT to identify febrile animals. Supplementation with LY increased neutrophils and monocytes, the 2 leukocytes most associated with an innate immune response. Furthermore, LY supplementation reduced haptoglobin concentration response in VB-challenged heifers, which indicates a reduction in the acute phase protein response. Despite the fact that LY supplementation appeared to mitigate the haptoglobin response induced by the VB challenge, LY supplementation did not ameliorate the impact of the VB challenge on DMI or BW. These results suggest that LY supplementation may be beneficial at reducing the catabolic effects associated with the acute phase response while increasing functionality of the innate immune system.

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