The rumen as a modulator of immune function in cattle


Modern dairy cows are typically fed grain-rich diets to fulfill their high energy requirements. Nonetheless, this feeding practice impairs chewing activity and production of buffering substances via saliva, while increasing volatile fatty acid (VFA) production in the rumen. This leads to subacute ruminal acidosis (SARA) (Plaizier et al., 2008; Kleen and Cannizzo, 2012). SARA is described as an intermittent drop of ruminal pH below 5.6 for longer than 3 hours/day (Gozho et al., 2005; Plaizier et al., 2008) or below 5.8 for longer than 5-6 hours/day (Zebeli et al., 2008). Although the bovine rumen can adapt to grain-rich diets by increasing the ruminal papilla epithelium surface, the rate of cellular aging can decrease to a level that induces parakeratosis and hyperkeratosis during sustained high-grain feeding. This compromises VFA absorption, reducing the pH and risking the onset of SARA (Zebeli and Metzler-Zebeli, 2012).

In a healthy rumen, the squamous multilayer epithelium acts as the main site for the absorption of key nutrients (i.e. VFA and electrolytes), and is highly selective to prevent simultaneous entry of microbes and luminal toxins into systemic circulation (Plaizier et al., 2018; Aschenbach et al., 2019). However, SARA can lead to a failure in the selective rumen epithelium barrier function, thereby enabling luminal immunogens to translocate into the blood supply © Burleigh Dodds Science Publishing Limited. 2020. All rights reserved.

and lymphatic system (Wu et al., 2016). More specifically, various luminal toxins such as endotoxins and biogenic amines seem to interfere with the epithelial constraint function by altering the structure and function of the tight junction barrier, thereby disrupting the integrity of epithelial cells and enabling their translocation changing cellular pathways (Berkes et al„ 2003).

There is a growing body of evidence that indicates that SARA leads to enhanced growth and lysis of Gram-negative bacteria (GNB) followed by the release of large amounts of cell-free lipopolysaccharides (LPS) (Nagaraja et al., 1978; Beutler and Rietschel, 2003; Plaizier et al., 2012) and biogenic amines (BA) such as ethanolamine and histamine (Dong et al., 2011). While some BA are considered as local inflammatory agents (Gozho et al., 2005), the gastrointestinal LPS, which is part of the outer membrane of GNB's cell wall, is an abundant and potentially pro-inflammatory molecule which has been investigated extensively in relation to immunity and disease (Emmanuel et al., 2008; Li et al., 2012a; Plaizier et al., 2012). Once gastrointestinal LPS enters into circulation, a pro-inflammatory cascade is triggered which is commonly known as a low-degree acute phase response (APR). This is characterized by moderate elevation of serum acute phase proteins (APP) with lipopolysaccharide-binding protein (LBP), serum amyloid A (SAA) and haptoglobin (Hp; Ceciliani et al.,

  • 2012) being the main markers of the ruminal LPS translocation in cattle (Iqbal,
  • 2013) . The SAA promotes clearance of LPS that enters circulation through LPS- SAA-lipoprotein complexes in hepatocytes and the bile (Ametaj et al., 2010a; Zebeli and Metzler-Zebeli, 2012).

On the other hand, LBP transports LPS to immune cells such as macrophages, monocytes and neutrophils to be detoxified (Gallay et al., 1994; Schumann et al., 1994). More specifically, since LBP carries LPS to macrophages, the membrane receptor-CD14 at the macrophage surface interacts with TLR-4 and myeloid differentiation factor 2 (MD-2) which signal activation of macrophages (Chow et al., 1999; da Silva Correia et al., 2001). These signals are recognized by the myeloid differentiation primary response gene 88 (MyD88) activating nuclear factor к-B (NFk-B) and pro-inflammatory cytokines, for example, tumor- necrosis factor (TNF)-a, interleukin (IL)-1(3, IL-6 and IL-8 (Erridge et al., 2002; Emmanuel et al., 2008; Ceciliani et al„ 2012; Plaizier et al., 2012). These pro- inflammatory cytokines activate inflammation and trigger fever, stress, low feed intake, lipolysis and other metabolic changes in host cattle (Zebeli and Metzler- Zebeli, 2012; Abaker et al., 2017).

Besides the systemic inflammatory responses caused by LPS, a higher nutrient requirement to support immune responses lowers the nutrients available for the synthesis of milk components (Dong et al„ 2011). Furthermore, when LPS is transported to the mammary gland via systemic circulation, the bacterial toxins may have harmful effects on the epithelial cell functions in the mammary gland (Dong etal.,2011). Indeed, it has been reported that dairy cows which fed high-grain/low-forage diets showed increased LPS-concentrations (determined using the limulus amebocyte lysate test) in mammary blood and epigenetic changes in the mammary tissues (Dong et al„ 2014) as well as increased LBP concentrations in milk (Khafipour et al., 2009a). The impact of SARA and the LPS in the gastrointestinal tract of dairy cows on immunity and metabolism has recently attracted attention with a number of comprehensive review articles (Plaizier et al., 2008,2012; Dong et al., 2011; Kleen and Cannizzo, 2012). However, current knowledge about the relationships between SARA, LPS and its resulting inflammation, systemic metabolism and the mammary immune system still needs to be summarized and reviewed. This review aims to update and synthesize recent research regarding the effect of SARA and the resulting increase in free rumen LPS on metabolism and health in cattle, with a special emphasis on the mammary gland of dairy cows.

Prevalence of subacute ruminal acidosis (SARA) in dairy herds

Subacute ruminal acidosis (SARA) is commonly viewed as a severe and prevalent health disorder of cattle, which particularly occurs during early and mid- lactating periods and causes substantial economic losses to the dairy industry (Garrett et al., 1997; Plaizier et al., 2008). Previous studies showed a prevalence of SARA of about 11 -27% in earlylactation and of 18-27% during mid-lactation (Garrett et al., 1997; Kleen et al., 2004; Tajik et al., 2009). Early lactating cows are predisposed to SARA when, during the transition period, the diet changes too abruptly from dry cow diets rich in forage to a lactating cow diet rich in starch (a transition which should take at least 4-5 weeks (Enemark, 2008; Humer et al., 2018a). Short adaptation to a grain-rich diet also increases the risk of SARA (Pourazad et al„ 2016). Another factor that may increase the risk of SARA in mid- lactating cows is high feed consumption, especially when diets are deficient in physicallyeffective neutral detergent fiber (Nordlund etal., 1995; Stone, 2004). Parity also seems to play a role, with primiparous cows having a greater risk of developing SARA when compared with multiparous cows (Humer et al„ 2015). This might be due to a less well-adapted rumen epithelium and microbiome, differences in feeding behavior and body weight and less experience on how to self-regulate the ruminal pH (Humer et al., 2018a).

According to field studies conducted in several countries, the overall prevalence of SARA in dairy farms ranges between 8-33% (Table 1). This difference might be due to several factors such as less intensive feeding in pasture-based systems predominating in Ireland and Australia, for instance, as well as differences in feeding and herd management. Higher SARA risks in larger herds may be dueto less intensive animal observation (Kleen etal., 2013). In general, SARA reduces the productivity of cattle operations (Nagaraja and

Table 1 Prevalence of subacute ruminal acidosis (SARA) in dairy cows of some countries

Prevalence (%)





Morgante et al. (2007)



Tajik etal. (2009)


United Kingdom

Atkinson (2013)



Enemark and Jorgensen (2001)



Kleen et al. (2013)



Kitkas et al. (2013)


The Netherlands

Kleen et al. (2009)



O'Grady et al. (2008)



Bramley et al. (2008)

Lechtenberg, 2007; Plaizier et al., 2008). It does so by reducing body condition, feed intake, milk production and milk energy efficiency (Yang and Beauchemin, 2006). Overall, losses due to SARA are reported to reach US$1.12/day per affected cow (Stone, 1999).

To minimize these economic losses, early detection of this disorder is crucial. Although SARA is considered as a sub-clinical disorder, SARA-affected cows might be recognized by para-clinical signs, such as reduced cud-chewing (Zebeli et al., 2010), milk fat depression (Zebeli and Ametaj, 2009), diarrhea, foamy feces (Nordlund and Garrett, 1994; Kleen et al„ 2003), and presence of undigested grain in feces (Enemark, 2008). On the other hand, the onset of SARA might also be related to other metabolic disorders (Aditya et al„ 2017), such as sudden death syndrome, fatty liver (Ametaj et al., 2005) and laminitis (Nocek, 1997). The reasons why SARA increases the risk of developing metabolic disorders in cattle have not yet been fully established. However, alterations to the rumen ecosystem, such as increase in GNB, and the immunological changes which result, have recently mentioned as playing a role (Plaizier et al., 2008, 2012; Dong et al., 2011; Kleen and Cannizzo, 2012; Zebeli and Metzler- Zebeli, 2012).

Rumen health, metabolic activity and disorders

Although activation of the APR is essential to eliminate agent(s) that cause inflammation and to reestablish homeostasis, prolonged inflammatory processes are associated with negative consequences for the host (Morris and Li, 2012; Lacetera, 2016). For instance, the higher energy requirements associated with activation of the APR result in lowered feed efficiency and might aggravate the negative energy balance of dairy cows, particularly during early lactation (Zebeli and Metzler-Zebeli, 2012; Lacetera, 2016). The APR can lead to changes in energy and lipid metabolism in different body tissues, including the mammary gland (Kushibiki et al., 2002; Khovidhunkit et al., 2004). Furthermore, it has been suggested that LPS and the activated APR may be involved in the development of multiple metabolic diseases, including displaced abomasum, fatty liver, liver abscesses, laminitis and downer cow syndrome (Nocek, 1997; Kleen et al., 2003; Plaizier et al., 2008; Zebeli et al., 2015). There is, however, currently limited information about the interaction between rumen health and the systemic metabolism in dairy cows. There has been little research into the consequences of increased concentrations of potentially toxic compounds in the rumen, for example, LPS and BA, and possible changes of metabolic pathways in systemic circulation in dairy cows.

Most studies investigating the effect of rumen fermentation disorders on systemic metabolism have focused on single metabolites, such as non- esterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), cholesterol and liver enzymes indicating liver tissue damage (Zebeli et al., 2011; Marchesini et al., 2013). Several studies report an association between rumen fermentation disorders and increased activity of liver enzymes (Humer et al., 2018b; Kroger et al., 2019). This corresponds with increased ruminal LPS and BA loads during high-grain feeding (Humer et al., 2018b). This suggests that these changes are caused by an enhanced clearance rate of LPS and other circulating toxins in the Kupffer cells of the liver (Lechowski, 1997; Marchesini et al., 2013). Studies have shown that, even when the ruminal pH increased, liver enzymes continued to increase during high-grain feeding of the cows for 2 weeks (Fig. 1). Thus, it can be assumed that cows require more time to restore liver health than to regulate ruminal pH, which can return to healthy levels almost within a few days after

Concentrations of liver enzymes

Figure 1 Concentrations of liver enzymes (aspartate aminotransferase (AST); glutamate dehydrogenase (GLDH)) in the blood of dairy cows that fed either a pure forage diet (Baseline) or a 65% concentrated diet causing subacute ruminal acidosis (SARA) during two consecutive weeks (SARA 1 and SARA 2). Different letters indicate differences among baseline, SARA 1 and SARA 2 at P < 0.05. Source: adapted from Humer et al.(2018b).

a high-grain diet (Kroger et al„ 2017; Khiaosa-ard et al., 2018). An increased activity of liver enzymes due to rumen fermentation disorders suggests a negative and potentially accumulative effect of high-grain feeding on LPS load and liver health, which might cause an impairment of overall health.

In recent years, metabolomic technologies have enabled the detection of multiple classes of metabolites reflecting changes in key metabolic pathways. These help to improve the understanding of interactions between nutrition, metabolism and health. Metabolomics is the quantitative analysis of all metabolites in an organism under specific conditions. Metabolites represent intermediates and end products of metabolic pathways. They are able to reflect physiological dysfunctions more rapidly than current biomarkers (such as APP or liver enzymes). Metabolomics may highlight earlier stages of metabolic disorders, helping to identify biomarkers of important cow diseases such as rumen fermentation disorders (Ametaj, 2010b).

Metabolomic technologies have been applied to rumen fluid samples in cows fed increasing levels of grain levels (Ametaj et al., 2010b; Saleem et al., 2012). More recently a metabolomics approach was also used to analyze blood samples taken from dairy cows that fed either a pure forage diet or receiving a high-grain diet (51% grains) which caused SARA (Humer et al., 2018b). Potentially toxic compounds, for example, BA and LPS, were analyzed in the rumen content and linked to changes in patterns of blood metabolic profiles using different data mining approaches (Humer et al., 2018b). Multivariate analyses indicated that cows experiencing SARA had elevated concentrations of ruminal LPS and BA (i.e. histamine, ethanolamine, isopropylamine, pyrrolidine, putrescine, cadaverine, spermidine), which accompanied significant changes in the blood metabolome. Decreases in phosphatidylcholines (PC; Fig. 2), lysophosphatidylcholines (lysoPC) and sphingomyelins were observed in particular. A decrease in PC has also been reported in the rumen fluid of cows that fed high-grain diets containing up to 45% barley grain (Saleem et al., 2012). This might be due to a decline in protozoa during rumen pH depression, which are the main source of PC in the rumen fluid (Jouany et al., 1988; Goad et al., 1998; Khafipour et al„ 2009). The decrease in PC probably also caused the decrease of lysoPC, because lysoPC are a hydrolysis product of PC(Hailemariam et al., 2014a). A decrease in plasma lysoPC has also been reported in cows receiving LPS from Escherichia coli (026:B6) intramammarily or experiencing diseases such as mastitis, metritis, retained placenta and laminitis (Hailemariam et al., 2014a; Humer et al., 2018c).

A further explanation for the decrease in PC might be the decrease in cholesterol, as PC are generally associated with cholesterol and triacylglycerols (Gruffat et al., 1996). As high levels of rapidlyfermentable carbohydrates are generally associated with a decreased production of precursors for cholesterol synthesis in ruminants (i.e. acetate; Liepa et al., 1978; Neubauer et al., 2018),

Concentrations of phosphatidylcholines

Figure 2 Concentrations of phosphatidylcholines (PC) with diacyl-residues (aa) in the blood of dairy cows that fed either a pure forage diet (Baseline) or a 65% concentrated diet causing subacute ruminal acidosis (SARA).

decreasing PC and lysoPC might be a common indicator in cows receiving high-grain diets. Sphingomyelins belong to the group of bovine phospholipids (Nilsson and Duan, 2006). This is supported by a strong positive association of PC, sphingomyelins and cholesterol, which were identified in one subcluster through multivariate and correlation analysis.

Figure 3 shows the effects on amino acids (AA). Decreases in arginine, citrulline, isoleucine, methionine, phenylalanine, tryptophan and tyrosine were observed while concentrations of glycine and serine increased. The decreasing effect of SARA-related feeding on AA concurs with previous studies reporting a pronounced decrease in cows receiving eitheran external £. Co//-LPS-challenge (Humer et al., 2018c) or experiencing one or several periparturient diseases (Hailemariam et al., 2014b). The underlying mechanism might be increased protein catabolism driven by the requirement for AA for immune cells, causing enhanced consumption of AA such as arginine or tryptophan (Le Floch et al., 2004; Hailemariam et al., 2014b). Only glycine and serine were enhanced in dairy cows experiencing SARA. An enhanced production of glycine and its precursor serine might be a way for cows to counterbalance SARA-associated inflammation and oxidative stress, as glycine has been reported to be protective against injuries and diseases due its antioxidant, anti-inflammatory, cytoprotective and immunomodulatory properties (Razak et al., 2017).

Multivariate analysis suggests a negative relationship between the concentration of several deleterious compounds in the rumen fluid (i.e. LPS, histamine, ethanolamine, pyrrolidine, and spermidine) and the concentration of AA, PC, lysoPC and sphingomyelins in the blood. This suggests that enhanced

Concentrations of amino acids in the blood of dairy cows that fed either a pure forage diet (Baseline) or a 65% concentrated diet causing subacute ruminal acidosis (SARA)

Figure 3 Concentrations of amino acids in the blood of dairy cows that fed either a pure forage diet (Baseline) or a 65% concentrated diet causing subacute ruminal acidosis (SARA).

release of toxic substances in the rumen in cows experiencing grain-induced SARA affect metabolic pathways for AA and lipid metabolism.

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