Anti-lipopolysaccharide antibody administration mitigates ruminal lipopolysaccharide release and depression of ruminal pH during subacute ruminal acidosis challenge in Holstein bull cattle.

The effects of anti-lipopolysaccharide (LPS) antibody on rumen fermentation and LPS activity were investigated during subacute ruminal acidosis (SARA) challenge. Eleven Holstein cattle (164 ± 14 kg) were used in a 3 × 3 Latin square design. Cattle were fed a roughage diet on days -11 to -1 (pre-challenge) and day 2 (post-challenge), and a high-grain diet on days 0 and 1 (SARA challenge). For 14 days, 0-, 2-, or 4-g of anti-LPS antibody was administered once daily through a rumen fistula. Ruminal pH was measured continuously, and rumen fluid and blood samples were collected on days -1, 0, 1, and 2. Significantly lower ruminal LPS activity on day 1 was observed in the 2- and 4-g groups than those in the 0-g group. In addition, significantly higher 1-hr mean ruminal pH on SARA challenge period (days 0 and 1) was identified in the 4-g group than in the 0-g group. However, rumen fermentation measurements (total volatile fatty acid [VFA], VFA components, NH3-N and lactic acid) and peripheral blood metabolites (glucose, free fatty acid, beta-hydroxybutyrate, total cholesterol, blood urea nitrogen, aspartate aminotransferase and gamma-glutamyl transferase) were not different among the groups during the experimental periods. Therefore, anti-LPS antibody administration mitigates LPS release and pH depression without the depression of rumen fermentation and peripheral blood metabolites during SARA challenge in Holstein cattle.

Various types of probiotic or microbial supplementations have been used to improve rumen fermentation and to prevent subacute ruminal acidosis (SARA) incidence in cattle [16, 20]. For example, administration of a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium, and Clostridium butyricum reduced the decrease in ruminal pH in Holstein calves fed a high-concentrate diet [16]. In addition, active dried Sacharomyces cerevisiae supplementation in calves produce more butyric and lactic acids as energy source during ruminal acidosis challenge [20]. Although probiotic or microbial supplementations may improve rumen fermentation, they were not able to entirely prevent the occurrence of SARA. Furthermore, strategies for controlling free ruminal lipopolysaccharide (LPS) have not been considered extensively.

In the cattle rumen, the bacterial community under the high-forage diet is composed of an approximately one-to-one ratio of Firmicutes and Bacteriodetes at a phylum level [11, 14]. However, higher acidity caused by a high-grain diet, as seen in ruminal acidosis or SARA, decreases the proportion of Bacteriodetes due to death or lysis of gram-negative bacteria, resulting in higher LPS activity in the rumen [1, 20]. Consequently, increased ruminal LPS can translocate to the blood-stream, triggering inflammatory [9] and acute-phase protein responses [4] in cattle. However, LPS bound to lipoproteins is removed from circulation by liver hepatocytes [7]. Therefore, LPS neutralization and related roles of liver cells are important in cattle fed a high-grain diet.

Previously, studies using LPS-binding peptides to neutralize LPS were performed in vitro using the phage display method [12] and peptide-bound beads [18], and in vivo studies were performed with mice [3, 22]. However, to the best of our knowledge, no research on anti-LPS antibody administration has been conducted in cattle despite the potential benefits of neutralizing and controlling rumen-induced LPS. Therefore, we investigated the effects of ruminal anti-LPS antibody administration on rumen fermentation and LPS activity during SARA challenge. We hypothesized that the use of an anti-LPS antibody in cattle fed a high-grain diet might mitigate the adverse effects of rumen-derived LPS activity.

MATERIALS AND METHODS

Anti-LPS antibody preparation

The anti-LPS antibody was produced under patented and proprietary procedures (EW Nutrition Japan., Gifu, Japan) as described elsewhere [23]. Briefly, the vaccine containing 1 ml of antigen (1 × 109 colony forming unit/g of inactivated whole Escherichia coli O139) with oil adjuvant was injected intramuscularly into egg-laying hens (Hy-Line W36), and the second injection was performed 8 weeks after the first injection. After the 2 weeks after the second injection, eggs were collected and stored at 4°C. The separated egg yolk from the collection was homogenized thoroughly, filtered to eliminate other components, and spray-dried (140 to 72°C) to prepare the product in a powder form. The result was 1 g of the product bound to 0.25 g of purified LPS from E. coli O111 as tested by in house ELISA method using the anti-E. coli O111:B4 LPS rabbit IgG capture antibody, anti-E. coli O111:B4 LPS guinea pig IgG primary antibody, and horseradish peroxidase conjugated anti-guinea pig IgG secondary antibody. We determined the amount of anti-LPS antibody based on the previously reported ruminal LPS concentration (up to 5 µg/ml) in growing Holstein steers (330 to 380 kg body weight) with approximately 100 l of rumen [13].

Animals and experimental design

The experimental protocol was approved by the Iwate University Laboratory Animal Care and Use Committee (A201453-1; Morioka, Japan). Eleven fistulated Holstein bull cattle (5–6 months of age) were used in a 3 × 3 Latin square design without a wash-out period. Cattle were fed a roughage (orchard and timothy mixed hay; 5.6–7.0 kg/day) diet during the first 11 days (days −11 to −1; pre-challenge), a high-grain (50% concentrate and 50% soybean flakes; 3.0–3.6 and 3.0–3.8 kg/day, respectively) diet for 2 days (days 0 and 1; SARA challenge), and then a roughage diet for 1 day (day 2; post-challenge) (Table 1). The cattle were administered 0 (control group), 2, or 4 g immunoglobulin yolk containing the anti-LPS antibody (EW Nutrition Japan) per head once daily through the rumen fistula for 14 consecutive days. The diets were supplied daily at 0800 hr and 1630 hr in two equal portions. Feed composition and amounts were based on the requirements of the Japanese Feeding Standard for Dairy Cattle, and all feeds offered to animals were consumed. No abnormal changes in body conditions and behaviors were observed daily throughout the study period.

Table 1.

Composition of the roughage and high-grain diets on percentage and dry matter (DM) bases

Item	Roughage diet	High-grain diet
Amount (%)
Orchard and timothy hay	100	0
Concentrate	0	50
Soybean flakes	0	50
DM basis1 (%)
TDN	60.9	80.5
CP	13.0	15.7
ADF	40.5	12.8
NDF	68.0	25.7
NFC	8.0	48.7
Ca	0.5	0.4
P	0.3	0.4

1TDN, total digestible nutrients; CP, crude protein; ADF, acid detergent fiber; NDF, Neutral detergent fiber; NFC, non-fiber carbohydrate.

Sampling and measurements

Ruminal pH was measured continuously every 10 min during the experimental days using a radio transmission system (YCOW-S; DKK-TOA, Shinjo, Japan), as described previously [17]. Rumen fluid and blood samples were collected, right before the morning feeding, at 0800 hr and 1400 hr on days −1, 0, and 1, and at 0800 hr on day 2 to analyze the total volatile fatty acid (VFA), VFA components, NH3-N and lactic acid concentrations, and LPS activity. The fluid samples were immediately filtered through two layers of cheesecloth and stored at −80°C until use. Blood samples were immediately centrifuged (1,500 × g, 15 min, 4°C) to separate the plasma and then preserved at −80°C until analysis.

For the VFA analyses, total VFA and individual VFAs (acetic, propionic, and butyric acids) were separated and quantified by gas chromatography (GC-2014; Shimadzu, Kyoto, Japan) as previously described [15]. For lactic acid analyses, the concentration in the supernatant was determined using a commercial F-kit (D-lactate/L-lactate) (J.K. International, Tokyo, Japan) as described previously [15]. To measure rumen LPS activity, a kinetic Limulus amebocyte lysate assay (Pyrochrome with Glucashield; Seikagaku, Tokyo, Japan) was used as previously described [10]. Serum and plasma were separated by centrifugation at 1,500 × g for 15 min at 4°C, and biochemical analysis was performed using an automated biochemistry analyzer (Accute, Toshiba, Tokyo, Japan).

Statistical analyses

The normality of the distributions of variables was assessed using the Shapiro–Wilk test. Significant differences among groups were evaluated using unpaired t-tests for normally distributed variables and the Mann–Whitney U-test for non-normal variables (Prism ver. 8.10; GraphPad Software, La Jolla, CA, USA). A mixed-model ANOVA (accounting for repeated measures), using time as a fixed effect, followed by Dunnett’s multiple comparison method was used to determine within-group differences. Significant differences were determined at a threshold of P<0.05.

RESULTS

Ruminal LPS activity

In all groups, no significant change in ruminal LPS activity was identified (P>0.10) during SARA challenge. However, significantly (P<0.05) lower LPS activities were identified on day 1 (1400 hr) in the 2-g and 4-g groups compared with the 0-g group (2.32 and 3.34 vs. 6.59 endotoxin unit ×103/ml, respectively) (Fig. 1).

Fig. 1.

Changes in the ruminal lipopolysaccharide (LPS) activity in Holstein bulls (n=11) after 0, 2, and 4 g of anti-lipopolysaccharide antibody were administered once daily. Days −2, −1, 0, 1, and 2 denote observations during the pre-challenge (day −2 and −1), subacute ruminal acidosis (SARA) challenge (day 0 and 1; gray square), and post-challenge (day 2) periods. *Denotes significant difference (P<0.05) compared with the 0-g group at that time point. Values represent mean ± SE.

Ruminal pH and VFA

The 24-hr mean ruminal pH was significantly (P<0.05) changed during SARA challenge period, and the durations and area under curves (for pH <5.6) were also significantly (P<0.05) changed during the same period (Table 2). The 24-hr minimum, mean, and maximum pH were significantly (P<0.05) decreased on day 0, 1, and 2 compared with day −1. On day 0, the 24-hr minimum ruminal pH was significantly (P<0.05) higher, and the duration under pH <5.6 and area under pH 5.6 were significantly (P<0.05) lower in the 4-g group compared with those in the 0-g group. Furthermore, the 1-hr mean ruminal pH was significantly (P<0.05) higher in the 4-g group during SARA challenge compared with the 0-g group (Fig. 2).

Table 2.

Changes in 24-hr mean ruminal pH, duration of time, and area under the curve (for pH <5.6) in Holstein bulls (n=11) administered 0, 2, and 4 g of anti-lipopolysaccharide antibody once daily

Item	Day −11	Day 0	Day 1	Day 2	SEM	P-value2
24-hr mean pH
Minimum
0 g	6.48	5.69a	5.15a	5.76a	0.12	<0.001
2 g	6.10	5.25a	4.93a	5.38a	0.11	<0.001
4 g	6.12	5.39ab	4.95a	5.30a	0.10	<0.001
Mean
0 g	6.12	4.96a	4.30a	4.99a	0.03	<0.001
2 g	6.52	5.92a	5.33a	6.08a	0.02	<0.001
4 g	6.44	5.99a	5.36a	6.07a	0.03	<0.001
Maximum
0 g	6.80	6.65a	5.93a	6.35	0.08	<0.001
2 g	6.81	6.80	5.95a	6.70	0.05	<0.001
4 g	6.71	6.60a	6.02a	6.86	0.05	<0.001
Duration of ruminal pH (min/day)
pH <5.6
0 g	0	650a	1,170a	615	83.2	<0.001
2 g	0	413a	1,078a	248	75.6	<0.001
4 g	2.5	148ab	1,120a	395a	55.7	<0.001
Area under curve (pH × min/day)
pH <5.6
0 g	0	27.4a	87.6a	34.3	5.99	<0.001
2 g	0	11.7	46.6a	9.90	4.32	<0.001
4 g	0	1.63ab	41.2a	14.4	4.44	<0.001

aDenotes significant difference (P<0.05) compared with day −1 in each group. bDenotes significant difference (P<0.05) between the 0- and 4-g groups at the same time point. 1Days −1, 0, 1, and 2 denote observations during the pre-challenge (day −1), high-grain diet challenge (day 0 and 1), and post-challenge (day 2) periods. 2Mixed effects model ANOVA, followed by Dunnett’s multiple comparison method, was used to determine within-group differences.

Fig. 2.

Diurnal changes in the 1-hr mean ruminal pH in Holstein bulls (n=11) after 0, 2, and 4 g of anti-lipopolysaccharide antibody were administered once daily. Days −2, −1, 0, 1, and 2 denote observations during the pre-challenge (day −2 and −1), subacute ruminal acidosis (SARA) challenge (day 0 and 1; gray square), and post-challenge (day 2) periods. Vertical arrows indicate feeding of a high-grain diet (0800 hr and 1630 hr). b,cDenote significant differences (P<0.05) between the 0- and 4-g groups and between the 2- and 4-g groups at that time point. Values represent mean ± SE.

The total VFA concentration, proportion of acetic, propionic, and butyric acids (0- and 4-g groups), and ratio of acetic acid-to-propionic acids (2- and 4-g groups), and NH3-N (2- and 4-g groups) and lactic acid (4-g group) concentrations were significantly (P<0.05) changed during SARA challenge (Table 3). The NH3-N concentration was significantly (P<0.05) decreased during the pre- (1400 hr on day −1 in the 0-g group) and SARA challenge in each group compared with pre-challenge (0800 hr).

Table 3.

Total volatile fatty acid (VFA), individual VFA proportions, acetic acid to propionic acid (A/P) ratio, NH3-N, and lactic acid concentrations in Holstein bulls (n=11) administered 0, 2, and 4 g of anti-lipopolysaccharide antibody once daily

Items	Day −11		Day 0		Day 1		Day 2	SEM	P-value2
	0800 hr	1400 hr	0800 hr	1400 hr	0800 hr	1400 hr	0800 hr
Total VFA (mmol/dl)
0 g	10.7	9.76	11.0	14.7a	14.5	17.0a	14.7a	0.83	<0.001
2 g	10.3	9.47	10.0	13.4a	15.6a	16.3a	13.5	0.68	<0.001
4 g	10.6	10.1	11.0	13.2	14.5a	17.8a	14.9a	0.60	<0.001
Acetic acid (%)
0 g	72.9	72.5	72.3	66.4a	56.5a	53.4a	52.0a	1.69	<0.001
2 g	72.9	72.5	72.6	67.6a	54.8a	51.6a	56.3a	1.79	<0.001
4 g	72.1	71.6	72.3	67.0a	57.5a	54.3a	59.4a	1.58	<0.001
Propionic acid (%)
0 g	17.9	17.6	18.8a	21.4a	22.9a	24.9a	22.2	1.12	0.033
2 g	18.3	18.0	18.9	20.9a	21.7a	22.8a	19.9	0.76	0.043
4 g	19.1	18.7	18.9	21.2a	23.2a	22.7	16.9	0.84	<0.001
Butyric acid (%)
0 g	7.13	8.15a	6.90	10.3a	17.9a	19.4a	22.1a	1.29	<0.001
2 g	6.64	7.60a	6.45	9.62a	20.6a	23.1a	21.0a	1.52	<0.001
4 g	6.99	8.06a	6.98	9.94a	16.5a	20.7a	21.1a	1.12	<0.001
A/P ratio
0 g	4.09	4.13	3.87	3.12	2.53	2.31	3.43	0.28	0.104
2 g	4.0	4.04	3.86	3.25a	2.63a	2.34a	3.26	0.20	0.015
4 g	3.83	3.85	3.84	3.17a	2.60a	2.51a	3.99b	0.21	0.014
NH3-N (mg/dl)
0 g	7.02	4.41	7.07	5.89	4.97	6.41	6.89	0.87	0.173
2 g	8.11	5.73a	7.78	6.06	3.59a	4.46a	6.22	0.85	0.006
4 g	6.28	3.92a	6.62	5.14	3.37	4.30	5.70	0.72	0.040
Lactic acid (g/l)
0 g	0.031	0.027	0.023	0.039	0.898	1.105	1.599	0.304	0.167
2 g	0.029	0.025	0.023	0.023	0.040	0.228	2.519	0.197	0.072
4 g	0.027	0.027	0.026	0.024	0.077	0.416	2.791	0.222	0.048

aDenotes significant difference (P<0.05) compared with day −1 in each group. bDenotes significant difference (P<0.05) between the 0- and 4-g groups at the same time point. 1Days −1, 0, 1, and 2 denote observations during the pre-challenge (day −1), high-grain diet challenge (day 0 and 1), and post-challenge (day 2) periods. 2Mixed effects model ANOVA, followed by Dunnett’s multiple comparison method, was used to determine within-group differences.

Blood metabolites

The concentrations of glucose (0- and 2-g groups), free fatty acid (0- and 2-g groups), beta-hydroxybutyrate (4-g group), and blood urea nitrogen (0- and 2-g groups) were significantly (P<0.05) changed during the SARA challenge period (Table 4). However, no significant differences (P>0.10) among group comparisons in the peripheral blood metabolites were found.

Table 4.

Biochemical analysis of peripheral blood in Holstein bulls (n=11) administered 0, 2, and 4 g of anti-lipopolysaccharide antibody once daily

Items3	Day −11		Day 0		Day 1		Day 2	SEM	P-value2
	0800 hr	1400 hr	0800 hr	1400 hr	0800 hr	1400 hr	0800 hr
GLU (mg/dl)
0 g	88.8	85.0	91.1	96.2	97.4	88.7	90.1	5.57	0.012
2 g	78.9	82.6	85.3	94.1	95.0	88.1	78.8	3.81	0.035
4 g	83.8	73.4	86.2	118.5	93.7	88.1	81.8	8.42	0.060
FFA (μEq/l)
0 g	119	139	154	74.0	57.4	53.6	83.6	24.8	0.031
2 g	101	93.5	135	67.1	62.4	56.9	64.1	18.0	0.013
4 g	86.3	81.4	84.0	65.0	59.3	53.2	59.4	10.6	0.209
BHB (µmol/l)
0 g	483	378	370	578	597	635	769	161	0.082
2 g	574	467	532	694	792	951	974	114	0.054
4 g	486	470	516	528	626	862	849	74.1	0.011
T-CHO (mg/dl)
0 g	75.9	70.9	68.2	64.5	60.2	64.5	63.6	5.03	0.175
2 g	72.8	72.7	69.0	68.4	65.2	64.0	63.6	3.69	0.119
4 g	80.5	74.6	72.5	70.3	68.4	71.1	70.5	7.35	0.461
BUN (mg/dl)
0 g	5.98	6.58	5.83	4.85	2.73	2.50	3.70	1.24	0.022
2 g	6.10	7.08	6.60	6.73	4.10	3.13	2.98	1.09	0.007
4 g	5.93	5.50	5.55	5.38	3.75	3.65	3.28	1.29	0.059
AST (U/l)
0 g	64.0	62.1	65.5	62.6	54.0	64.7	67.5	4.40	0.441
2 g	61.0	57.0	56.2	55.3	97.5	111	59.3	17.3	0.544
4 g	56.6	54.0	57.0	64.8	54.6	54.1	59.9	5.03	0.518
GGT (U/l)
0 g	16.4	15.1	16.0	16.3	15.3	15.2	16.4	1.13	0.761
2 g	17.7	13.7	17.0	16.2	14.2	18.1	17.9	2.81	0.852
4 g	15.6	13.3	15.6	18.6	13.9	15.9	16.2	1.89	0.775

1Days −1, 0, 1, and 2 denote observations during the prechallenge (day −1), high-grain diet challenge (day 0 and 1), and postchallenge (day 2) periods. 2Mixed effects model ANOVA, followed by Dunnett’s multiple comparison method, was used to determine within-group differences. 3GLU, glucose; FFA, free fatty acid; BHB, β-hydroxybutyrate; T-CHO, total cholesterol; BUN, blood urea nitrogen; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase.

DISCUSSION

In the present study, SARA, a condition characterized by ruminal pH <5.6 for an extended period [9], was successfully induced during the SARA challenge. Moreover, the anti-LPS antibody did not affect rumen fermentation and blood metabolites but did suppress ruminal LPS release during SARA challenge. For example, significantly lower LPS activities observed on day 1 (1400 hr) in the 2-g and 4-g groups compared with the 0-g group were likely due to high binding affinity of anti-LPS antibody to ruminal LPS. Furthermore, the total VFA concentration and major VFA components (acetic, propionic, and butyric acids) showed the same direction of change during SARA challenge, and these results were consistent with the general features of a high-grain diet in cattle studies [8, 19, 21]. In addition, NH3-N and lactic acid concentrations among the groups were changed in the same direction. Therefore, the administration of anti-LPS antibody did not cause significant changes in the rumen fermentation and blood metabolite profiles, preferably leading the selective suppression of ruminal LPS activity in the present study.

Once rumen-derived LPS translocates to the circulation, activated Kupffer cells release more pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-6 in the systemic circulation, triggering secretion of APPs such as LBP, HP, and SAA [7, 21]. However, administration of anti-LPS antibody alleviated ruminal pH depression and ruminal LPS suppression during SARA challenge in the present study, the effects of anti-LPS antibody administration on pro-inflammatory cytokines of the peripheral blood and transcriptome expression of the liver were unknown. Therefore, further studies need to reveal the effects of anti-LPS antibody on systemic immune responses, and liver functions due to rumen derived LPS in SARA cattle.

In the present study, an administration of anti-LPS antibody alleviated ruminal pH depression during SARA challenge. Cattle in the 4-g group showed significantly lower 1-hr mean pH on days 0 and 1, simultaneously with significantly lower duration of ruminal pH and area under curve (pH <5.6) in the 4-g group those compared with the 0-g group. The duration of time and area under the pH curve (for 5.6) on day 0 was likely due to significantly higher 24-hr minimum pH compared with that of the 0-g group. However, we could not provide plausible evidence that the 1-hr mean ruminal pH was less depressed in the anti-LPS antibody administration groups, and temporally higher minimum ruminal pH on day 0 in the 0-g group than the 4-g group or higher 1-hr mean ruminal pH on day 1 (1500 hr) in the 4-g group than the 2-g group due to limited information on physiological responses to lowered ruminal pH [2, 5]. In addition, the anti-LPS antibody showed a high binding affinity to LPS that might have possible impacts on living Gram-negative bacteria in accordance with other studies using avian-derived polyclonal antibodies against Fusobacterium necrophorum and Streptococcus bovis in crossbred steers [6] and anti-LPS-enriched colostrum in a mouse model [3]. Therefore, further studies are required to clarify the detailed regulatory mechanism of anti-LPS antibody in the rumen, including its potential effect on physiological responses and bacterial community structures.

In conclusion, anti-LPS ruminal administration mitigated LPS release and pH depression without any depression of rumen fermentation and blood metabolites among the 0-, 2-, and 4-g groups. Further studies are required to elucidate the effects of anti-LPS antibody on systemic immune responses, and liver transcriptomic adaptations, and the regulatory mechanism of ruminal pH through analysis of the rumen bacterial community.

CONFLICT OF INTEREST

The authors have nothing to disclose.