Impacts of polyclonal antibody preparations from avian origin as a feed additive to beef cattle: immune responses during the step-up transition diets

Abstract

This study investigated the effects of feeding an avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (LPS; 40%, 35%, and 25% of the preparation, respectively) on immune responses (haptoglobin [Hp], serum amyloid A [SAA], rectal temperature [RT], leukocyte counts, and expression of cell adhesion molecules cluster of differentiation [CD] CD11b, CD14, and CD62L) of beef steers during a 21-d step-up adaptation to a high-grain diet. Eight ruminally cannulated Angus crossbred beef steers (658 ± 79 kg of BW) were assigned in a cross-over design and transitioned from a diet containing bermudagrass hay (Cynodon dactylon (L.) Pers.) ad libitum plus 0.45 kg/d of molasses with 0 (CON) or 3 g of PAP to a high-grain diet. Transition consisted of three 7-d steps of increased inclusion of cracked corn (35%, 60%, and 82% of the diet dry matter for STEP1, STEP2, and STEP3, respectively). On each transition day and 7 d after STEP3 (STEP3-7d), RT was obtained every 3 h for a total of 24 h, whereas blood was collected on days 0, 1, and 3, relative to diet transition. There were no effects of PAP inclusion in any of the blood parameters (P > 0.11). However, a tendency for day effect (P = 0.10) was observed for concentrations of Hp, which were greater on days 3 and 7 vs. day 0 relative to the second diet transition (STEP2). Plasma concentrations of SAA were greater on days 1, 3, and 7 compared to day 0 during STEP1 (P = 0.01), while during STEP2 and STEP3, SAA concentrations increased (P < 0.01) from day 0 to 3. During STEP2, PAP steers tended to have lower (P = 0.08) RT than CON steers. Neutrophil and monocyte counts were the least during STEP3 (P < 0.01), whereas expression of CD11b and CD62L was the least through forage feeding (P < 0.01). Concentration of starch in the diet was correlated to all the variables tested (P ≤ 0.01), except for the percentage of B cells (P = 0.22). Yet only ruminal pH, RT, monocyte, and neutrophil counts presented strong correlation coefficients. In conclusion, the step-up transition from forage to high-grain diets triggered systemic inflammation in beef steers as observed by increased plasma concentrations of Hp, SAA, and expression on adhesion molecules in leukocytes. However, feeding polyclonal antibody preparations against S. bovis, F. necrophorum, and LPS did not provide benefits to mitigate inflammation.

Introduction

Diets rich in rapidly fermentable carbohydrates, such as starch, increase the risk of acidotic events in beef cattle (NASEM, 2016), with a variety of studies associating the increased amount of grain in the diet with systemic inflammation in cattle (Gozho et al., 2005, 2007; Plaizier et al., 2018). The systemic inflammation is proposed to be initiated by dietary-induced damage to the gut mucosa and translocation of immunogenic compounds into circulation, such as lipopolysaccharide (LPS; Horadagoda et al., 1999; Kvidera et al., 2017). Grain-based diet challenges increase the concentrations of the acute phase proteins (APP), such as serum amyloid A (SAA) and haptoglobin (Hp) in peripheral blood of cattle (Gozho et al., 2007; Emmanuel et al., 2008). Even though the inflammatory response is needed to maintain homeostasis, chronic inflammation has negative consequences for animal health (Elsasser et al., 2008) and performance (Johnson, 1997). Ruminal bacteria, such as Streptococcus bovis and Fusobacterium necrophorum, respond to an increased availability of starch by boosting its growth rates in grain-fed animals (Nagaraja and Lechtenberg, 2007), rising the risk of disease in cattle. Gradually transitioning beef cattle to high-grain diets is an important strategy used to reduce the intensity of metabolic diseases; however, the transition period is still the time when cattle encounter the greatest risk of ruminal acidosis (Fulton et al., 1979).

Feed additives, such as ionophores, are extensively used to enhance cattle performance by promoting alterations in ruminal microbial populations and fermentation (DiLorenzo et al., 2006), especially during high-grain feeding. Nevertheless, other technologies, such as polyclonal antibody preparations (PAP), have been investigated as a possible tool to ameliorate the effects of high-grain diets in cattle health and performance. Research using PAP against S. bovis and F. necrophorum was effective in increasing ruminal pH of beef steers (DiLorenzo et al., 2006) and heifers (Blanch et al., 2009) fed high-grain diets, while PAP against S. bovis improved feed efficiency of feedlot beef steers (DiLorenzo et al., 2008). In dairy cows, milk production was enhanced when PAP against LPS was provided even though cow health status did not change (Ibarbia et al., 2014). However, to our knowledge, this is the first study evaluating the effects of PAP on immune response of beef steers during transition from forage to a high-grain diet.

Therefore, we hypothesize that feeding PAP against S. bovis, F. necrophorum, and LPS during the transition from forage to high-grain diets would reduce the diet-induced stress in beef steers during the 21-d step-up process. Our objective was to evaluate the effects of feeding PAP as a strategy to minimize systemic inflammation in beef cattle during a 21-d step-up transition from forage to high-grain diets.

Materials and Methods

The Institutional Animal Care and Use Committee of the University of Florida (protocol #201810277) approved all procedures for the experiments conducted at the North Florida Research and Education Center (NFREC; Marianna, FL).

Polyclonal antibody preparations

The PAP against S. bovis (ATCC 9809), F. necrophorum (ATCC 27852), and LPS from Escherichia coli O157:H7 and bacteria from the genus Salmonella (LPS; 40%, 35%, and 25% of the preparation, respectively) are produced under patent and proprietary procedures (Camas Inc., Le Center, MN); thus, refer to DiLorenzo et al. (2006, 2008) for portions of the production process. The powder preparation used in the current study comprised the whole egg (egg white and yolk) and contained immunoglobulin Y (IgY), immunoglobulin M, and immunoglobulin A. The molasses with PAP provided in the current experiment was analyzed before the start of the study by specific ELISA test plates (Corning Inc., Corning, NY) using the same proportion that was fed to steers (3 g of PAP in 0.450 kg of as fed liquid molasses) to monitor antibody concentrations. It was detected 0.003 mg/g of IgY in the liquid molasses and PAP mix.

Experimental design, animals, and treatments

Eight ruminally cannulated Angus crossbred steers (658 ± 79 kg of body weight [BW]) were used in a cross-over design with two periods of 36 d each plus 26 d of washout within periods. Steers were randomly assigned to receive 0 (CON; 4 steers per period) or 3 g/d of PAP (PAP; 4 steers per period) that was individually fed using 0.45 kg/d of liquid molasses as a carrier during the transition from a forage (bermudagrass hay [Cynodon dactylon (L.) Pers.]) to a high-grain diet through a 21-d step-up process.

From d −7 to 0, steers were fed only bermudagrass hay (56% total digestible nutrients [TDN] and 13.9% crude protein [CP] in a dry matter [DM] basis) ad libitum. From day 0 to 14, steers received 0.45 kg/d of liquid molasses with or without the addition of PAP and ad libitum bermudagrass hay; feeding PAP 14 d before the diet transition was needed to ensure adequate delivery of PAP in the rumen during the diet change. Chemical composition of the molasses used was (DM basis): 7.8% CP, 1.3% crude fat, 15% ash, 76% TDN, 1.23% Ca, 0.10% P, 0.45% Mg, 4.99% K, 0.127% Na, 1.17% S, 107 mg/kg Fe, 15 mg/kg Zn, 18 mg/kg Cu, 12 mg/kg Mn, and 1.3 mg/kg Mo. The molasses provided had 76% DM on as fed basis.

The diet transition consisted of three steps (STEP1, STEP2, and STEP3) that lasted 7-d each, in which the inclusion of cracked corn increased gradually (35%, 60%, and 82% of the diet DM, respectively) in replacement of cottonseed hulls (Table 1).

Table 1.

Ingredients and nutritional composition (dry matter basis) of experimental diets fed during the step-up transition

	STEP 1	STEP 2	STEP 3
Ingredients, % DM
Cottonseed hulls	52	27	5
Corn grain, cracked	35	60	82
Bermudagrass hay	5	5	5
Cottonseed meal	2	2	2
Liquid supplement1	5	5	5
Limestone	1	1	1
Nutritional composition, % DM
DM, %	89	91	89
CP	11.1	12.4	12.3
aNDF2	58.3	35.5	17.3
ADF2	38	23.2	9.7
TDN	69	75	81
Starch	21.6	35	60.5

¹Molasses-based supplement containing urea, vitamins, and minerals. Molasses-based supplement containing (DM basis): 76% DM, 7.8% CP, 1.3% crude fat, 15% ash, 76% TDN, 1.23% Ca, 0.10% P, 0.45% Mg, 4.99% K, 0.127% Na, 1.17% S, 107 mg/kg Fe, 15 mg/kg Zn, 18 mg/kg Cu, 12 mg/kg Mn, and 1.3 mg/kg Mo.

²Alpha amylase neutral detergent acid (aNDF) and acid detergent fiber (ADF).

Ruminal pH, rectal temperature, blood, and feed sampling

Basal samples of ruminal fluid and rectal temperature (RT) were obtained during the day of each diet transition and STEP3-7d (days 14, 21, 28, and 35) before the molasses feeding and diet change (0 h), continuing every 3 h for a period of 24 h. Ruminal fluid was collected from four representative sites within the rumen to obtain a sample of digesta and squeezed through 4 layers of cheesecloth. The pH of ruminal fluid was immediately measured using a manual pH meter (Corning Pinnacle M530, Corning, Inc., Corning, NY). RT was measured using a veterinary rectal thermometer for large animals.

Samples of STEP1, STEP2, and STEP3 diets were collected immediately after delivery on days 14, 21, and 28, respectively. Following collection, samples were dried at 55 °C for 48 h in a forced-air oven, ground in a Willey mill to pass a 2-mm sieve and sent to a commercial laboratory for chemical analyses (Table 1).

Blood samples (approximately 10 mL) were collected via jugular venipuncture into sodium-heparin containing tubes (158 USP; Vacutainer, Becton Dickinson, Franklin Lakes, NJ) for the collection of plasma before the molasses feeding and diet change (0 h) of each transition day and on days 1 and 3, relative to the start of each step. Blood samples were immediately placed on ice following collection and then centrifuged for 15 min at 4,000 × g at 4 °C. After centrifugation, plasma was transferred into polypropylene vials (12 × 75 mm; Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA), and stored at −20 °C for further analysis. Plasma concentration of Hp was determined in duplicate samples using a biochemical assay evaluating the haptoglobin–hemoglobin complex by the estimation of differences in peroxidase activity (Cooke and Arthington, 2013). Plates were read at 450 nm in a microplate spectrophotometer (Multiskan Go, Thermo Fisher Scientific). Inter- and intra-assay coefficients of variation of Hp were 4.4% and 2.8%, respectively. SAA was determined using a commercial kit (Tridelta Diagnostics Inc., Morris Plains, NJ; cat. nos. TP-802) and a microplate spectrophotometer at 520 nm (Fisherbrand UV/VIS AccuSkan GO Spectrophotometer, Thermo Fisher Scientific Inc., Hampton, NH). Intra- and inter-assay coefficients of variation for SAA were 3.6% and 3.7%, respectively.

On day 14 of the experiment before PAP feeding (day 0 relative to diet transition) and day 3 relative to each diet change, samples of heparinized whole blood were collected to performed flow cytometry analysis as described by Cangiano et al. (2019) for monocyte, neutrophil, and B cells (CD21⁺) counts and cell adhesion molecules cluster of differentiation (CD) CD11b, CD14, and CD62L. Samples were analyzed using an Accuri C6 digital analyzer flow cytometer (Becton Dickinson Biosciences, San Jose, CA) with B cells, monocytes, and neutrophils being analyzed based on their size and granularity in the density cytogram. Data acquisitions of the total amount of cells per sample were analyzed using Flowjo software v10.1 (Single Cell Analysis Software, LLC, OR).

Statistical analyses

Data were analyzed as a cross-over design using the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, version 9.4), with steer as the experimental unit. The model included fixed effects of period, order, treatment, time, and treatment × time (hour or day of the study) interaction; random variables for all the analyses included effects of steer within period, order, and treatment. Covariance structures for repeated measures were based upon the smallest Akaike Information Criterion values and used steer within order, period, and treatment as the subject. The PROC CORR procedure of SAS was used to detect correlations existing among percentage of starch in the diet, ruminal pH, SAA, Hp, RT, and leukocytes. Significance was set at P ≤ 0.05, and tendencies were considered when 0.05 < P ≤ 0.10.

Results

Plasma concentrations of Hp and SAA were not affected by treatment × day interaction or treatment during the diet transition (P ≥ 0.43; Table 2). However, a tendency for day effect was observed during STEP2 (P = 0.10; Table 2). Hp concentrations tended to be greater (P = 0.10) on days 3 and 7 vs. day 0, relative to diet transition (0.34 and 0.36 vs. 0.11 ± 0.071 mg/mL, respectively). Blood plasma concentration of Hp gradually increased from STEP1 to STEP3 (P = 0.01; days 0 to 24 of the study; Figure 1), in which Hp concentrations were greatest on day 31 (STEP3) and least on day 14 (before diet change; 0.51 vs. 0.01 ± 0.09 mg/mL, respectively).

Table 2.

Plasmatic concentrations of haptoglobin and serum amyloid A of cannulated beef steers fed free-choice step-up diets that contained increased concentrations of cracked corn¹ plus 0.45 kg of molasses that was individually fed containing or not 3 g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40%, 35%, and 25% of the preparation, respectively)

	Treatment			Days relative to transition					P-value
	PAP	CON	SEM	0	1	3	7	SEM	Trt	Day	Trt × Day
Haptoglobin, mg/mL
STEP1	0.11	0.11	0.011	0.09	0.12	0.10	0.11	0.018	0.65	0.75	0.93
STEP2	0.29	0.26	0.093	0.11y	0.21xy	0.34x	0.36x	0.071	0.94	0.10	0.99
STEP3	0.49	0.34	0.149	0.36	0.41	0.47	0.37	0.149	0.45	0.81	0.43
Serum amyloid A, μg/mL
STEP1	31.5	27.3	6.19	9.8b	36.6a	33.4a	37.7a	8.65	0.64	0.01	0.34
STEP2	62.0	50.2	10.10	37.7c	66.5b	76.5a	43.6bc	10.50	0.43	0.01	0.76
STEP3	50.3	56.6	14.60	43.6b	62.2a	61.7a	46.0ab	11.90	0.76	<0.01	0.15

¹Diets consisted of gradual inclusion of cracked corn from 0 (Bermudagrass hay only) to 35%, 60%, and 82% STEP1 (days 14 to 21), STEP2 (days 21 to 28), and STEP3 (days 28 to 35), respectively.

^a,bWithin a row, means with different superscripts differ, P ≤ 0.05.

^x,yWithin a row, means with different superscripts tend to differ, P > 0.05 and ≤ 0.10.

Figure 1.

Plasmatic concentrations of haptoglobin of Angus crossbred steers fed free-choice step-up diets that contained increased concentrations of cracked corn (35%, 60%, and 82%; STEP1 [days 14 to 21], STEP2 [days 21 to 28], and STEP3 [days 28 to 35], respectively) plus 0.45 kg of molasses that was individually fed containing or not 3 g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40%, 35%, and 25% of the preparation, respectively). Effect of day on haptoglobin (P = 0.011; SEM = 0.09) but not treatment × day (P = 0.81) or treatment (P = 0.81) were detected. Within STEP, means without a common superscript (a–b) differ (P ≤ 0.05).

Plasma concentrations of SAA were greater on days 1, 3, and 7 compared to day 0, relative to diet transition during STEP1 (P = 0.01; Table 2; 36.6, 33.4, and 37.7 vs. 9.8 ± 8.65 μg/mL, respectively), whereas in STEP2, SAA concentrations were the greatest on day 3 relative to transition (P = 0.01; 76.5 vs. 37.7, 66.5, and 43.6 ± 10.5 μg/mL for days 3, 0, 1, and 7, respectively). During STEP3, SAA increased on days 1 and 3 post-diet transition compared with days 0 and 7 (62.2 and 61.7 vs. 43.6 and 46.0 ± 11.9 μg/mL, respectively). Within steps, SAA concentrations were greatest during STEP2 (P < 0.01; from day 21 to 25 of the study; 76.5 ± 10.0 μg/mL) compared with STEP1 and STEP3 (day 14 to 21 and day 27 to 35 of the study; Figure 2) and least on day 14 of the study (9.8 ± 10.0 μg/mL).

Figure 2.

Plasmatic concentration of serum amyloid A (SAA) of Angus crossbred steers fed free-choice step-up diets that contained increased concentrations of cracked corn (35%, 60%, and 82%; STEP1 [days 14 to 21], STEP2 [days 21 to 28], and STEP3 [days 28 to 35], respectively) plus 0.45 kg of molasses that was individually fed containing or not 3 g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40%, 35%, and 25% of the preparation, respectively). Effect of day on SAA (P < 0.001; SEM = 10.0) but not treatment × day (P = 0.65) or treatment (P = 0.96) were detected. Within STEP, means without a common superscript (a–d) differ (P ≤ 0.05).

A treatment × hour effect was detected for RT of beef steers on STEP1 (P = 0.03; Figure 3A). Steers that received PAP had greater RT on h 0 compared with CON (38.9 vs. 38.3 ± 0.13 °C, respectively), whereas during STEP2, PAP steers tended to have lowered RT than CON steers (P = 0.08; 39.2 vs. 39.6 ± 0.12 °C, respectively). Overall RT gradually increased from STEP1 to STEP3 (P ≤ 0.05; Figure 4; 38.8, 39.4, 39.8 ± 0.13 °C for STEP1, STEP2, and STEP3, respectively).

Figure 3.

Rectal temperature (RT) within the first 24-h post-diet transition of Angus crossbred steers fed free-choice step-up diets that contained increased concentrations of cracked corn (35%, 60%, and 82%; STEP1, STEP2, and STEP3, respectively) plus 0.45 kg of molasses that was individually fed containing or nor 3 g of polyclonal antibody preparations (PAP) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40%, 35%, and 25% of the preparation, respectively). Effect of treatment × hour interaction post-transition was detected on RT during STEP1 (A; P = 0.03; SEM = 0.158). A tendency effect of treatment during STEP2 (B; P = 0.08; SEM = 0.253), an effect of hour on STEP3 (C; P = 0.005; SEM = 0.206) and STEP3-7d (D; P < 0.001; SEM = 0.184) were detected. Within hours, means without a common superscript differ (P ≤ 0.05).

Figure 4.

Rectal temperature of Angus crossbred steers fed free-choice step-up diets that contained increased concentrations of cracked corn (35%, 60%, and 82%; STEP1 [day 14], STEP2 [day 21], STEP3 [day 28], and STEP3-7d [day 35], respectively) plus 0.45 kg of molasses that was individually fed containing or not 3 g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40%, 35%, and 25% of the preparation, respectively). Effect of day (P < 0.001; SEM = 0.13) but not treatment × day (P = 0.12) or treatment (P = 0.48) were detected. Within day, means without a common superscript (a–c) differ (P ≤ 0.05).

There were no effects of treatment × step or treatment on leukocyte profile (P ≥ 0.13; Table 3). However, an effect of step (P ≤ 0.02) was observed in all the leukocytes measured. Increasing the amount of grain in the diet, boosted the expression of CD62L and CD11b in B cells, monocytes, and neutrophils, whereas monocytes and neutrophil counts (mL of blood) were reduced when more grain was added to the diet (P < 0.01).

Table 3.

Mean fluorescence intensity of expression of cluster of differentiation (CD) 11b and 62L by peripheral blood monocyte, neutrophil, and CD21⁺ of cannulated beef steers fed free-choice step-up diets that contained increased concentrations of cracked corn plus 0.45 kg of molasses that was individually fed containing or not 3 g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40%, 35%, and 25% of the preparation, respectively)¹

	Treatment		Diet						P-value
Cell type2	PAP	CON	SEM	Forage	STEP1	STEP2	STEP3	SEM	Trt	Diet	Trt × Diet
Monocyte, mL blood	742.3	801.8	101.24	1,536.8a	684.2b	420.1c	409.3c	112.54	0.671	<0.01	0.816
MonocyteCD62L, log MFI	2.8	2.8	0.17	2.8b	2.6b	2.5b	3.4a	0.23	0.965	<0.01	0.575
MonocyteCD11b, log MFI	3.6	3.5	0.03	3.4c	3.7a	3.6a	3.5b	0.04	0.279	<0.01	0.141
MonocyteCD14, log MFI	4.3	4.2	0.05	4.4a	4.5a	3.9b	4.2b	0.09	0.870	<0.01	0.495
CD21+CD62L, MFI	1,600	1,458	492	613b	1,917a	1,734a	1,853a	427.6	0.841	<0.01	0.890
CD21+CD11b, MFI	902	1,169	393	370b	936ab	1,580a	1,256a	373.0	0.640	<0.01	0.276
CD21+, %	33.2	31.7	1.68	28.3b	35.8a	35.6a	29.9b	2.17	0.520	<0.01	0.654
Neutrophil, mL blood	1,021	1,444	204	2,983a	1,047b	503bc	325c	400.1	0.168	<0.01	0.107
NeutrophilCD62L, log MFI	3.7	3.7	0.03	3.35c	3.8b	3.9a	3.9a	0.04	0.727	<0.01	0.411
NeutrophilCD11b, log MFI	3.6	3.4	0.03	3.4c	3.7a	3.6ab	3.5bc	0.07	0.291	<0.01	0.757

¹Diets consisted of gradual inclusion of cracked corn from 0 (Bermudagrass hay only) to 35%, 60%, and 82% STEP1 (days 14 to 21), STEP2 (days 21 to 28), and STEP3 (days 28 to 35), respectively.

²Blood leukocytes were analyzed by flow cytometry based on cell size, granularity, and absence of CD14 expression. Monocytes were identified by size, granularity, and presence of CD14, whereas neutrophils were indicated by size and granularity only. B cells (CD21⁺) were identified by size, granularity, and absence of CD14. Abundance of each protein was indicated by the median fluorescence intensity.

^a,bWithin a row, means with different superscripts differ, P ≤ 0.05.

Starch in the diet was correlated to all the variables tested (P ≤ 0.01; Table 4), except for the percentage of B cells (P ≤ 0.22). Starch was negatively correlated with ruminal pH (P ≤ 0.01; r = −0.66), monocyte (P < 0.01; r = −0.59), and neutrophil counts (P < 0.01; r = −0.587), while positively correlated with RT (P < 0.01; r = 0.53). The APP, SAA and Hp, were positively correlated with starch (r ≥ 0.24) and correlated to each other (P ≤ 0.01; r = 0.38).

Table 4.

Pearson correlation coefficients (top number) and P-value (bottom number) for starch in diet and blood immune markers of cannulated beef steer fed free-choice step-up diets that contained increased concentrations of cracked corn (35%, 60%, and 82%; STEP1 [days 14 to 21], STEP2 [days 21 to 28], and STEP3 [days 28 to 35], respectively) plus 0.45 kg of molasses that was individually fed containing or not 3 g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40%, 35%, and 25% of the preparation, respectively)

Item1	Starch	SAA, μg/mL	Hp, mg/mL	RT, °C	Ruminal pH	CD21+, %	Monocyte, mL blood
SAA, μg/mL	0.336
	<0.001
Hp, mg/mL	0.275	0.383
	0.001	<0.001
RT, °C	0.528	0.149	−0.033
	<0.001	0.165	0.759
Ruminal pH	−0.662	−0.266	−0.131	−0.476
	<0.001	0.041	0.223	<0.001
CD21+, %	−0.181	−0.255	−0.018	−0.114	−0.245
	0.217	0.078	0.901	0.495	0.143
Monocyte, mL blood	−0.596	−0.281	−0.112	−0.468	0.694	−0.324
	<0.001	0.050	0.448	0.003	<0.001	0.024
Neutrophil, mL blood	−0.587	−0.332	−0.137	−0.420	0.623	−0.148	0.655
	<0.001	0.024	0.35	0.008	<0.001	0.315	<0.001

¹Number of observation per variable: Serum amyloid A (SAA; n = 143), haptoglobin (Hp; n = 140), rectal temperature (RT; n = 88), ruminal pH (n = 90), CD21⁺ (n = 48), monocyte (n = 48), and neutrophil (n = 48).

Discussion

A variety of studies have associated the increased amount of grain in the diet with systemic inflammation in cattle (Gozho et al., 2005, 2007; Plaizier et al., 2018). The systemic inflammation is proposed to be initiated by dietary-induced damage to the gut mucosa, ruminal and intestinal, and translocation of immunogenic compounds, such as LPS, into circulation (Horadagoda et al., 1999). To cause an inflammatory or acute phase response (APR), free circulating LPS must combine with LPS binding protein (LBP), and the resulting compound should bind to cell receptors, such as CD14, TLR-4, and MD2 (Tomlinson and Blikslager, 2004). The binding of the LPS–LBP complex to immune cell receptors promotes the secretion of pro-inflammatory cytokines, such as TNF-α, IL-1, and IL-6, that will further stimulate the synthesis of SAA and Hp in hepatocytes (Plaizier et al., 2018). To our knowledge, this was the first study that investigated the effects of PAP on immune responses of beef cattle during the step-up diet transition.

There were no differences in plasma concentrations of SAA and Hp when PAP was fed. Despite the lack of differences between treatments, the transition from forage to a high-grain diet increased plasma concentrations of SAA and Hp, confirming that our model was successful in inducing systemic inflammation as SAA and Hp are used as inflammatory markers in cattle (Alsemgeest et al., 1994). SAA is recognized to be directly involved in the clearance of endotoxin (Cabana et al., 1999) and SAA stimulates leukocytes to secrete cytokines (Patel et al., 1998). Hp inhibits cyclooxygenase and lipoxygenase activities, which are enzymes that catalyze reactions harmful to body tissues (Saeed et al., 2007). In agreement with our study, Gozho et al. (2005) reported a rise in concentrations of serum Hp (from 0.53 to 1.40 mg/mL) and SAA (from 38 to 163 μg/mL) in ruminally cannulated Jersey steers when a diet containing 76% of concentrate (50% ground wheat and 50% ground barley) was offered. In healthy cattle, circulating concentrations of Hp and SAA are minimal or nonexistent (Heegaard et al., 2000). In the present study, concentrations of SAA and Hp were least when steers were consuming forage only, which indicates that steers were not in an inflammatory state at the beginning of the study. SAA concentrations were the greatest when steers were consuming 65% of cracked corn (STEP2), while plasmatic concentrations of Hp peaked when 82% of cracked corn (STEP3) in the diet was fed. Jacobsen et al. (2004), challenging Holstein dairy cows with intravenous injections of increasing doses of LPS, reported that SAA concentrations started to increase and peaked before Hp levels, and Hp levels remained elevated for a longer time compared to SAA. Usually, SAA is more sensitive, presenting a faster response to a stimulus, while Hp is known for its slower and longer response (Alsemgeest et al., 1994; Horadagoda et al., 1994, 1999), thus explaining the different peaks observed in the study.

The inflammatory innate immune response is primarily mediated by monocytes, macrophages, and neutrophils, which can recognize LPS pathogen-associated molecular patterns (Daibert et al., 2020). Reduction in circulating leukocyte counts can be caused by increased cell demand during metabolic disorders and infectious diseases (Roland et al., 2014). While the provision of PAP did not impact peripheral blood leukocytes, the diet transition from forage to high-grain diets reduced monocyte and neutrophil counts and increased cell expression of CD11b and CD62L in monocytes, B cells, and neutrophils. Compared with other species, there is a lack of storage of mature neutrophil in adult cattle, which results in an initial reduction in number of neutrophils rather than a neutrophilic reaction during early (first 24 to 48 h) inflammatory responses (Jones and Allison, 2007; Roland et al., 2014). In addition, neutrophils have a short life span and cannot undertake repeated phagocytosis (Day and Schultz, 2014), contributing to reduce blood cell count during inflammation. Reduction in neutrophil and monocyte have been observed during acute inflammatory response in cattle (Jones and Allison, 2007; Roland et al., 2014). The results observed in the current study are consistent with the expected delay in the production of leukocytes because of the time needed to generate cells in the bone marrow after infection. The percentage of B cells increased with the addition of grain in the diet. LPS is known to cause B cell activation (Parekh et al., 2003), resulting in increased circulating naive B cells, which have greater expression of CD62L (Morrison et al., 2010).

This further corroborates with the increased expression of the adhesion molecules CD11b and CD62L. Adequate expression of CD62L and CD11b is necessary for a successful immune response against bacterial infections as these molecules facilitate adherence of leukocytes to endothelium and subsequent migration into infected tissue (Perkins et al., 2001). Upregulation of peripheral blood leukocyte CD11b is used as a marker of inflammation in cattle (Poindexter et al., 2020), whereas downregulation in the expression of CD62L and CD11 is associated to impaired migration of neutrophils (Meglia et al., 2001). CD14 is an important cell adhesion molecule for toll-like receptors and Gram-negative recognition, phagocytosis, and production of pro-inflammatory cytokines (Cangiano et al., 2019; Abdelsalam et al., 2020). Expression of CD14 in monocytes was downregulated when steers were consuming the diets containing 60% and 82% inclusion of corn (STEP2 and STEP3) compared with when steers offered 100% forage or 35% corn (STEP1). In viral in vitro cell infections, Abdelsalam et al. (2020) observed downregulation of CD14 in monocyte when cells were infected with high virulent strains of bovine viral diarrhea virus (BVDV), whereas no effects on expression of CD14 were observed when monocytes were infected with low strains of BVDV. Cangiano et al. (2019) found increased expression of CD14 in monocyte cell surface when beef heifers were challenged with intravenous LPS injections. Nevertheless, different factors contribute to changes in expression of CD14, and its expression can be upregulated in immature cell lines or downregulated in more mature monocytes (Ziegler-Heitbrock and Ulevitch, 1993). Thus, the reduction in expression of CD14 in monocytes in the present study could be associated with more circulating mature monocytes, as observed by the decrease in monocyte counts.

Typically, in addition to increased hepatic production of SAA and Hp, the APR is accompanied by a fever reaction (Hughes et al., 2014), which can destroy bacteria and accelerate the proliferation of immune cells (Carroll and Forsberg, 2007). The febrile response involves alteration of the body temperature in the hypothalamus, with pro-inflammatory cytokines, IL-1, IL-6, and TNF-α, regulating the fever response through the induction of prostaglandin E2 (Carroll and Forsberg, 2007). RT was measured to understand if a febrile response was associated with high-grain feeding. Corroborating with the APP and leukocytes data, RT of the steers increased with the inclusion of grain, with RT being greater than the proposed average non-febrile RT for beef cattle of 38.5 °C (Herring, 2014). Feeding PAP to beef steers during the diet transition period reduced RT; however, it is not clear if PAP acted direct to minimize the immune response to high-grain diets or if PAP reduced total tract digestibility of starch as observed in previous research (Silva et al., 2020) and consequently RT due to lowered fermentation in the hindgut.

In this study, during the diet transition, APP, RT, monocyte, and neutrophil counts were positively correlated with starch concentration in the diet. However, an increase in RT is not always observed during high-grain feeding (Gozho et al., 2007). Zebeli et al. (2012) performed a meta-analysis and described a relationship between the level of concentrate in the diet with ruminal pH and endotoxin (r² = 0.27), combined with plasma Hp (r² = 0.19) and SAA levels (r² = 0.46). The strong correlation between starch and ruminal pH is expected as it is known that grain feeding can increase concentration of volatile fatty acids in rumen fluid, which can accumulate and reduce ruminal pH. As previously mentioned, high-grain feeding can trigger an inflammatory state through translocation of free endotoxins from rumen into the bloodstream, contributing to increased APP. Additionally, it is important to highlight the contribution of the hindgut acidosis to cause systemic inflammation as it shares similarities with ruminal acidosis (accumulation of acids, reduction in pH, and shifts in microbial populations; Gressley et al., 2011). Indeed, many symptoms before attributed to rumen acidosis is now also accredited to the hindgut leakage, but the lack of studies designed to isolate the different effects of acidosis in rumen and hindgut makes it difficult to determine the exact contribution of each gastrointestinal section in the general syndrome (Sanz-Fernandez et al., 2020). It is possible that the benefits of providing PAP during diet transition could have been limited to rumen only and not being enough to reach and mitigate the acidotic conditions encountered in the hindgut, therefore, limiting its effectiveness on the host immune responses.

In conclusion, the step-up transition from forage to high-grain diets triggered systemic inflammation in beef steers as observed by increased plasma concentrations of Hp, SAA, and expression of adhesion molecules in leukocytes. Feeding polyclonal antibody preparations against S. bovis, F. necrophorum, and LPS did not provide benefit to mitigate inflammation.

Glossary

Abbreviation

ADF

acid detergent fiber

APP

acute phase proteins

APR

acute phase response

BW

body weight

CD

cluster of differentiation

CON

control

CP

crude protein

DM

dry matter

Hp

haptoglobin

IgY

immunoglobulin Y

LPS

lipopolysaccharide

LBP

LPS binding protein

NDF

neutral detergent fiber

PAP

polyclonal antibody preparations

RT

rectal temperature

SAA

serum amyloid A

TDN

total digestible nutrients

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.