Journal of Animal Science, 2020, Vol. 98, No.

8, 1–9

doi:10.1093/jas/skaa248
Advance Access publication August 6, 2020
Received: 4 June 2020 and Accepted: 30 July 2020
Board Invited Reviews

Board Invited Reviews

Ruminal acidosis, bacterial changes, and
lipopolysaccharides
Hugo F.  Monteiro and Antonio P. Faciola1
Department of Animal Sciences, University of Florida, Gainesville, FL 32611

Corresponding author: afaciola@ufl.edu

1

ORCiD numbers: 0000-0003-0935-6233 (A. P. Faciola).

Abstract
Acute and subacute ruminal acidosis (SARA) are common nutritional problems in both beef and dairy cattle. Therefore,
the objective of this review is to describe how ruminal Gram-negative bacteria could contribute to the pathogenesis
of ruminal acidoses, by releasing lipopolysaccharides (LPS; a component of their cell wall) in the ruminal fluid. When
cattle consume excessive amounts of highly fermentable carbohydrates without prior adaptation, normal fermentation
become disrupted. The fermentation of these carbohydrates quickly decreases ruminal pH due to the accumulation
of short-chain fatty acids and lactate in the rumen. As a consequence, ruminal epithelium may be damaged and
tissue function could be impaired, leading to a possible translocation of pathogenic substances from the rumen into
the bloodstream. Such changes in fermentation are followed by an increase in Gram-positive bacteria while Gram-
negative bacteria decrease. The lyses of Gram-negative bacteria during ruminal acidosis increase LPS concentration
in the ruminal fluid. Because LPS is a highly proinflammatory endotoxin in the circulatory system, past studies have
raised concerns regarding ruminal LPS contribution to the pathogenesis of ruminal acidosis. Although animals that
undergo these disorders do not always have an immune response, recent studies showed that different Gram-negative
bacteria have different LPS composition and toxicity, which may explain the differences in immune response. Given the
diversity of Gram-negative bacteria in the rumen, evaluating the changes in the bacterial community during ruminal
acidosis could be used as a way to identify which Gram-negative bacteria are associated with LPS release in the rumen.
By identifying and targeting ruminal bacteria with possible pathogenic LPS, nutritional strategies could be created to
overcome, or at least minimize, ruminal acidosis.

Key words:  Bacteroidetes, cattle, dysbiosis, endotoxin, Fibrobacteres, Gram-negative bacteria

  

Ruminal Acidosis in Cattle behavior), change the fermentation pattern toward nonfibrous
carbohydrates (NFC) fermentation (Owens et al., 1998; Nagaraja
Acute and subacute ruminal acidoses (SARA) have long been
and Titgemeyer, 2007; Miller-Cushon and DeVries, 2017). In the
described as a digestive disorders that affect beef and dairy cattle
rumen, these carbohydrates are quickly fermented into short-
(Owens et al., 1998; Nagaraja and Titgemeyer, 2007; Plaizier et al.,
chain fatty acids (SCFA) and lactate, and the accumulation of
2008). The transition from forage-based diets to concentrate-
these acids lowers ruminal pH to below normal fermentation
based diets, containing greater levels of rapidly fermentable
conditions (e.g., pH < 5.6), which can last for hours (Nagaraja
carbohydrates (e.g., transition to lactation or feedlot), or even
and Titgemeyer, 2007). This disruption negatively affects animal
the abrupt consumption of these carbohydrates (e.g., sorting

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 2 | Journal of Animal Science, 2020, Vol. 98, No. 8

Abbreviations investigating the origins of these endotoxins and the toxicity

EPM extracellular polymetric matrix of the LPS that could potentially affect the animal. Therefore,
EU endotoxin units the goal of this review is to describe the most current data on
GIT gastrointestinal tract bacterial changes during ruminal acidosis in order to elucidate
IL interleukin which bacterial groups could be contributing to the ruminal LPS
LAB lactic acid-producing bacteria increase. Understanding the origins of LPS could potentially
LAU lactate-utilizing bacteria indicate the toxicity of this endotoxin and the correlation of LPS
LPS lipopolysaccharides with ruminal acidosis.
NFC nonfibrous carbohydrates
PUFA polyunsaturated fatty acids
SARA subacute ruminal acidosis
Ruminal Lipopolysaccharide Origins
SCFA short-chain fatty acids
SFA saturated fatty acid Despite dietary, genetic, and environmental effects, different
TLR4 Toll-like receptor 4 ruminant species have been reported to have a core ruminal
microbiome composed of a mixture of Gram-positive and Gram-
negative bacteria (Henderson et  al., 2015). In cattle, the core
health and production; however, the pathogenesis of ruminal
ruminal microbiome has been reported to comprise 45% of all
acidosis is still not well understood.
ruminal bacteria, from which five out of six phyla are Gram-
A well-researched theory is that the increased acid load
negative bacteria (Xue et  al., 2018). During ruminal acidosis,
in the rumen may damage ruminal epithelium (Owens et  al.,
the bacterial community composition of the rumen changes,
1998; Nagaraja and Titgemeyer, 2007; Aschenbach et  al.,
and the concentration of Gram-positive bacteria increase, while
2019). According to Owens et  al. (1998), the animal undergoes
Gram-negative bacteria decrease (Andersen et al., 1994a; Li et al.,
a metabolic acidosis when the base excess in the blood is
2012b). The decrease in Gram-negative bacteria during acidosis is
decreased, due to ionized acid absorption by the rumen
followed by the accumulation of residues from bacterial lysis in
epithelium, and not corrected through blood pH homeostasis.
the ruminal fluid. As shown in Figure 1, Gram-negative bacteria
The accumulation of acids in the rumen also leads to an
contain LPS in their outer membrane and that is released during
increase in the ruminal osmotic pressure, which could cause
bacterial growth, division, and lysis; although bacterial lysis is
ruminal epithelium damage (Owens et  al., 1998; Nagaraja and
normally the main contributor to free LPS (Petsch and Anspach,
Titgemeyer, 2007; Aschenbach et  al., 2019). Tissue function
2000; Yaron et al., 2000), the diet associated with ruminal acidosis
becomes impaired when the ruminal epithelium is damaged
may increase the turnover rate of bacteria, and thus, intact cells
during ruminal acidosis, which increases the chances of
may also contribute to free LPS during ruminal acidosis as well
pathogenic substances translocating into the bloodstream
(Plaizier et  al., 2014). Thus, during ruminal acidosis, LPS from
(Plaizier et  al., 2012; Aschenbach et  al., 2019). Bacteria and
Gram-negative bacteria accumulates in the ruminal fluid and
bacterial endotoxins (lipopolysaccharides [LPS]) are believed to
may be toxic to the animal (Plaizier et al., 2012), such as the LPS
be able to translocate into the bloodstream in these situations,
from Escherichia coli that is known to evoke an immune response
potentially impacting animal health and production (Plaizier
when in the bloodstream (Nagaraja et  al., 1978b; Raetz et  al.,
et al., 2012, 2018; Aschenbach et al., 2019).
1991). Interestingly, the ruminal LPS has not been characterized
Although the diversity of the microbial population in
yet and its toxicity is not well understood. Therefore, in order to
the rumen is well noted, the literature is scarce of studies
understand the toxicity level of ruminal Gram-negative bacteria,

Figure 1.  Gram-negative bacterial cell-wall structure with emphasis in the lipopolysaccharide (LPS) presence in the outer membrane. Toxicity is mainly associated with
the lipid A composition. Courtesy of Yen-Ming Hsu, AB Biosciences, Inc. Boston, Massachusetts, USA.

it is important to first understand how the pattern of ruminal
LPS concentration is in animals under SARA and acute ruminal
acidosis, and how they are linked to the bacterial changes in
both disorders.
Subacute ruminal acidosis
SARA, while more prevalent in lactating dairy cows due to
their high NFC intake in order to meet energy demands for
lactation, it is also common in beef cattle consuming finishing
diets (Kleen et al., 2003; Aschenbach et al., 2011; Li et al., 2012a).
The excessive intake of NFC leads to an imbalance between the
production and clearance of ruminal SCFA. When the production
of SCFA surpasses the rate of absorption or clearance through
the flow of ruminal content out of the rumen, the ruminal
pH drops and stays below the optimal threshold (below pH of
5.6) for at least 3 h (Gozho et al., 2005; Penner et al., 2011). The
clinical signs are reduced feed intake and milk production,
milk fat depression, diarrhea, and laminitis. Thus, except for
laminitis, the symptoms of SARA are almost imperceptible on
a daily basis, which makes it a difficult problem to be identified
compared with acute acidosis (Kleen et al., 2003; Plaizier et al.,
2008, 2012).
The cause of these effects remains unclear; however, ruminal
LPS has been suggested to be one of the factors contributing to
these effects (Nagaraja and Titgemeyer, 2007; Plaizier et al., 2012;
Aschenbach et al., 2019). Because of the dietary changes during
the transition to SARA, the ruminal pH is later reduced, which
increases Gram-negative bacterial lysis. Besides that, substrates
for Gram-negative bacteria are reduced and the growth of some
Gram-negative bacteria may be impaired. Some research also
suggests that Gram-positive bacteria produce bacteriocins
against a wide variety of bacteria, including many Gram-
negative bacteria (Martinez et al., 2013), and that Gram-negative
bacteria themselves produce microcins against other Gram-
negative bacteria (Sassone-Corsi et al., 2016). These substances
could also increase the lysis of Gram-negative bacteria, given
that SARA is marked by an increase in Gram-positive bacteria.
Therefore, these dietary changes increase bacterial lysis in
animals under SARA, and the lysis of Gram-negative bacteria
leads to the release of cell-wall components, most notably LPS.
Past studies that evaluated LPS in the rumen reported LPS
concentration in endotoxin units (EU), which 1 EU contains 100
pg of LPS; thus, as one Gram-negative bacterium contains 10–
15
g of LPS, around 105 bacteria are necessary to produce 1 EU
(Raetz et al., 1991). In an induced SARA experiment, Gozho et al.
(2007) reported that cows with SARA had increased free ruminal
LPS from 24,547 EU to 128,825 EU/mL compared with animals
without SARA, although LPS was not detected in the blood.
In a similar study, Li et al. (2012b) reported an increase in free
LPS in both ruminal (from 10,405 to 168,391 EU/mL) and cecal
digesta samples (16,508 to 118,522 EU/g of wet cecal digesta).
Interestingly, some studies did report increases of LPS both
in the rumen and in the blood, although the reasons are still
unknown (Khafipour et  al., 2009; Zhao et  al., 2018). Khafipour
et al. (2009) showed that ruminal LPS increased from 28,184 to
107,152 EU/mL in a comparison of control dairy cows to those
with induced SARA, and these animals also had an increased
plasma LPS from <0.05 to 0.52. Later, Zhao et al. (2018) reported
that when ruminal LPS increased from 30,768 to 130,589 EU/
mL between control dairy cows and those with SARA, and
plasma LPS increased from <0.005 to 0.21 EU/mL, these animals
had an upregulated cascade of proinflammatory responses.
According to Zhao et  al. (2018), the expression and production
of tumor necrosis factor-α, interleukin-1ß (IL-1ß), and IL-6 were
Monteiro and Faciola  |  3
upregulated in the rumen epithelium due to high ruminal LPS
concentration, and that these could be one of the reasons for
animals experiencing rumenitis while with SARA.
Along with an increase of LPS concentration, studies have
shown that animals with SARA have reduced richness and
diversity in both ruminal and fecal bacteria (Mao et  al., 2013;
Plaizier et al., 2017a, 2017b), with some reporting an increase in
E. coli in both the hindgut and feces (Plaizier et al., 2017b). Plaizier
et  al. (2017a) reported that among the bacterial phyla with
more than 0.1% of relative abundance in the rumen, two Gram-
positive phyla (Firmicutes and Actinobacteria) increased with
SARA, while two Gram-negative phyla decreased (Cyanobacteria
and Verrucomicrobia); the phylum Firmicutes had the greatest
increase, from 22.4% in control cows to 35.7% in those with SARA.
However, one of the reasons for the increase in the phylum
Actinobacteria for animals with SARA was the increase in its
family Bifidobacteriaceae. In the rumen, the main genus of this
family is Bifidobacterium, which is a major lactic acid producer in
the rumen and is known to increase its abundance during SARA
and acute ruminal acidosis (Nagaraja and Titgemeyer, 2007;
Plaizier et  al., 2017a). Besides that, the genus Bifidobacterium is
also known to produce most of the bacteriocins against Gram-
positive and Gram-negative bacteria, suppressing bacterial
growth and increasing bacterial lysis in nonruminant studies
(Martinez et al., 2013). The bacteriocin activity from Bifidobacteria
has been reported to comprise a wide pH range (pH 2 to 10), but
most of them have enhanced activity at pH 4 to 7, which some
of those that target Gram-negative bacteria reported to have
adequate activity at pH 4.8 to 5.5 and temperature 37 to 40  °C
(Martinez et  al., 2013), conditions similar to SARA. In Plaizier
et  al. (2017a), animals with SARA also had decreased bacterial
diversity in the feces, and all bacterial phyla that decreased
in the feces of those animals with SARA were Gram-negative
(Tenericutes, Proteobacteria, Cyanobacteria, Fibrobacteres,
Verrucomicrobia), while the phylum Actinobacteria of Gram-
positive bacteria increased as in the rumen.
Plaizier et  al. (2017a) also showed that that Firmicutes
and Bacteroidetes together represented 76.9% and 94.4% of
the ruminal and fecal microbial population, respectively. The
difference from the ruminal to fecal samples was mainly due to
Proteobacteria, which had a relative abundance of 13.1% in the
rumen and only 0.67% in the feces. Most notably, E. coli, which
LPS has been used as a model for inflammation studies, is one
of the main bacteria from Proteobacteria. Interestingly, these
diets with increased grain content in order to cause SARA also
increase the passage rate of digesta out of the rumen and allow
more fermentable substrates to reach the hindgut (Van Soest,
1994). Increased grain fermentation in the hindgut may cause
similar effects of ruminal acidosis, which increases epithelial
damage and may allow the entry of LPS to the circulation
(Gressley et al., 2011). The LPS concentration has been reported
to increase in the hindgut (from 16,508 to 118,522 EU/g of wet
digesta) when grain-based diets are fed to induce SARA (Li et al.,
2012b). Thus, Proteobacteria could be an important source of
free LPS in the hindgut of animals experiencing SARA, as this is
a major Gram-negative bacteria in the hindgut and it is almost
absent in the feces (Mao et al., 2015; Plaizier et al., 2017a).
Mccann et al. (2014) pointed out that changes in the ruminal
bacterial population of cows right after parturition are also likely
to be associated with SARA, as these animals also have a sudden
intake of highly fermentable carbohydrates after calving. Similar
to Plaizier et  al. (2017a), Wang et  al. (2012) reported that after
calving, cows had an increase of the ruminal Gram-positive
phylum Firmicutes (55% at day −21 vs. 68% at day +21 after
 4 | Journal of Animal Science, 2020, Vol. 98, No. 8
calving), and a decrease in the ruminal Gram-negative phylum
Bacteroidetes (14% at day −21 to 5% at day +21 after calving).
The increase in Firmicutes was mainly due to an increase in
Anaerovibrio lipolytica, Streptococcus bovis, and Lactobacillus spp.
The last two bacteria are ruminal species responsible for the
fermentation of starch and sugar mainly into lactate, thus
associated with SARA and acute ruminal acidosis (Nagaraja
and Titgemeyer, 2007). However, the decrease in Bacteroidetes
was mainly related to a decrease in the family Rikenellaceae.
A  similar decrease in Rikenellaceae was also observed in beef
cattle transitioning from bermudagrass hay diets (crude protein
[CP]  =  11.2%; neutral detergent fiber [NDF]  =  67%) to grazed
winter wheat diets (CP  =  19.9%; NDF  =  44%), in an experiment
investigating the transition from low- to high-quality forages
(Pitta et  al., 2010). This study also showed that the latter diet,
which was a more digestible diet (in vitro dry matter [DM]
digestibility  =  57.2% vs. 80.3%), led to a decrease of the genus
Rikenella, which is from Rikenellaceae family reported to
decrease after the transition to lactation. The decrease and lysis
of these Gram-negative bacteria are another possible sources of
ruminal free LPS.
Recently, Dai et al. (2019) in a study evaluating LPS dosing into
dual-flow continuous culture fermenters in order to simulate
SARA conditions showed that LPS dosing decreased bacterial N
compared with normal fermentation conditions, which could
represent a decrease in microbial mass. Interestingly, this study
showed that LPS dosing increased the relative abundance of some
bacteria related to starch digestion and pH reduction in ruminal
fermentation (Anaeroplasma, Ruminobacter, Succiniclasticum,
Succinimonas, and Succinivibrio), despite those bacteria being
Gram-negative. In a follow-up study, Dai et  al. (2020) reported
that LPS may be used as a substrate for the growth of bacteria
related to acidosis (S.  bovis, Succinivibrio dextrinosolvens,
Lactobacillus ruminis, and Selenomonas ruminantium), and that
when LPS is present in a medium containing mixed bacteria
from ruminal fermentation, bacterial genes associated with the
biosynthesis of LPS were also increased.
Acute ruminal acidosis
Acute ruminal acidosis has been reported to be more prevalent
in beef than dairy cattle, specifically in feedlot cattle consuming
high-grain diets (Nagaraja et  al., 1998; Owens et  al., 1998;
Nagaraja and Titgemeyer, 2007). The cause of acute ruminal
acidosis is similar to SARA, however, at a greater level of highly
fermentable carbohydrate intake (mainly starch). Usually, high-
producing dairy cows consume around 30% starch in their
diet DM (60:40 forage to concentrate ratio), while beef cattle in
feedlot diets may have up to double the starch concentration of
dairy rations (10:90 forage to concentrate ratio). The transition
to these diets can cause the onset of acute ruminal acidosis,
as the ruminal microbial population of these animals are not
adapted to such high concentrations of starch (Owens et  al.,
1998; Nagaraja and Titgemeyer, 2007).
Lactic acid-producing bacteria (LAB), specifically S.  bovis,
rapidly ferment starch and glucose to produce lactate in the
rumen (Hungate, 1966; Nocek, 1997), reaching a doubling time
of just a few minutes (McAllister et  al., 1990). Lactate starts to
accumulate because lactate-utilizing bacteria (LAU), particularly
Megasphaera elsdenii and S.  ruminantium (predominant LAU),
replicate slowly and take weeks to considerably increase their
population in the rumen (Nocek, 1997; Russell and Rychlik,
2001). Consequently, lactate accumulates quickly, thus dropping
ruminal pH. Because of a resistance to more acidic pH and due
to bacteriocin-mediated response to S.  bovis, Lactobacillus spp.
thrive in these conditions and further reduce the ruminal pH
(pH < 5.0; Wells et  al., 1997). Although some authors consider
acute ruminal acidosis when pH is below the threshold of 5.2
(Nagaraja and Titgemeyer, 2007), whenever ruminal pH becomes
that low, it is because ruminal lactate concentration is greater
than normal (>5.0  mM) and animals are undergoing acute
ruminal acidosis. Animals experiencing acute ruminal acidosis
have been reported to reach ruminal lactate concentrations from
50 to 120  mM (Nagaraja et  al., 1998; Nagaraja and Titgemeyer,
2007). Clinical signs of acute ruminal acidosis include loss of
appetite, ruminitis, laminitis, polioencephalomalacia, and death
(Nocek, 1997; Nagaraja et  al., 1998; Nagaraja and Titgemeyer,
2007).
Unlike SARA, acute ruminal acidosis has been reported to
depress SCFA concentration (SCFA < 100  mM), and this may
happen due to a few reasons: 1) ruminal pH in animals with acute
ruminal acidosis may impair fermentation by some bacterial
communities until pH returns to normal conditions (Nagaraja
et  al., 1998); 2)  SCFA concentration may decrease because of
the influx of liquid from the osmotic changes caused by acute
acidosis (Owens et al., 1998); and 3) because the pKa of SCFA is
around 4.9, most of the SCFA in ruminal acidosis conditions will
be unprotonated, which should, in contrast, increase the SCFA
absorption by the rumen epithelium (Bergman, 1990). Whether
the major reason for lower ruminal SCFA concentration is
because of depressed SCFA production, the ruminal influx of
liquid diluting the SCFA concentration, or the increase in SCFA
absorption, Harmon et  al. (1985) reported that lactate isomers
have different absorption rates by the ruminal epithelium.
The isomer l-lactate has considerably greater absorption than
d-lactate, and this makes the accumulation of d-lactate one of
the possible causes of low ruminal pH (Harmon et al., 1985).
In acute ruminal acidosis, the association of Gram-negative
bacterial LPS and acidosis was first reported by Mullenax et al.
(1966) and Nagaraja et al. (1978b), which suggested that ruminal
bacteria contained an endotoxin similar to those found in
E. coli and Salmonella typhosa. Mullenax et al. (1966) injected the
bacterial endotoxin extracted from the rumen in sheep and beef
cattle and reported an animal toxicity similar to that of E. coli.
Nagaraja et  al. (1978b) showed that although similar to E.  coli,
ruminal bacterial endotoxins had lesser toxicity when tested in
embryos, mice, rabbits, and Limulus lysate. The ruminal bacteria
in the latter study was extracted from the rumen of cattle fed
hay or grains, containing both live and dead bacteria. The yield
of total endotoxin was greater for hay-fed cattle, as these diets
promote the growth of fibrolytic bacteria that are mostly Gram-
negative. In a subsequent study (Nagaraja et al., 1978a), animals
under acute acidosis had an increased concentration of ruminal
free endotoxins by 18 times in 12  h. In that study, an in vitro
experiment also showed that the decrease in Gram-negative
bacteria was not associated with an increase in endotoxins, and
thus, the release of endotoxins from intact cells seemed to have
increased during acidotic conditions. The authors suggested
that such an increase in free endotoxins may be due to other
factors, such as low ruminal pH; however, the increase in free
endotoxins may be related as well to the large increase in Gram-
positive bacteria during this period (e.g., bacteriocins).
Similar to SARA, LPS concentration has been reported to
increase due to an increase in Gram-negative bacterial lysis
(Nagaraja and Titgemeyer, 2007). However, animals under
acute acidosis conditions have been reported to have free LPS
concentrations of up to 408 EU/mL of ruminal fluid (Andersen
et al., 1994a), which is considerably greater compared with SARA.
Some studies also showed that the effects of acute ruminal

acidosis may be more associated with the LPS toxicity to the
animal than the LPS presence in the rumen itself (Dougherty
et  al., 1975; Aiumlamai et  al., 1992; Andersen et  al., 1994b),
as these studies did not report an immune response as that
observed by Mullenax et  al. (1966) and Nagaraja et  al. (1978b).
The lack of LPS toxicity in some studies is another reason for
reviewing bacterial changes in these animals and thus possibly
tracking the origins of LPS during acute ruminal acidosis.
Earlier research reported that increases in genera of bacteria
associated with lactate production in the rumen and possible
association with acute acidosis (Nagaraja and Titgemeyer, 2007).
These bacteria were Bifidobacterium, Butyrivibrio, Eubacterium,
Lactobacillus, Mitsuokella, Prevotella, Ruminobacter, Selenomonas,
Streptococcus, Succinimonas, and Succinivibrio. Besides the
bacteriocins produced by Bifidobacterium spp. explained earlier
in the Subacute ruminal acidosis section, LAB bacteriocins are
some of the main bacteriocins characterized to date, as those
are of great importance for the food industry sector (Cotter et al.,
2005; Perez et  al., 2014; Silva et  al., 2018). In the rumen, most
of the characterized bacteriocins are produced by the phylum
Firmicutes, specifically Clostridiales and Lactobacillales,
yet most of the LAB that increase in the rumen under acute
ruminal acidosis have shown to produce bacteriocins (Russell
and Mantovani, 2002; Azevedo et al., 2015). The bacteriocins in
the rumen, although not completely understood, indicate that
the increase in LAB and Gram-positive bacteria during acute
ruminal acidosis may be associated with an increase in Gram-
negative bacterial lysis, as explained earlier.
Past reviews on ruminal acidosis have emphasized the
importance of animal adaptation to high concentrate diets in
order to decrease the chances of acute ruminal acidosis (Owens
et  al., 1998; Nagaraja and Titgemeyer, 2007). This is a common
practice in the beef cattle industry, and it is known as a step-up
protocol, a method that allows LAU (mainly M.  elsdenii and
S.  ruminantium) to sufficiently increase their population in the
rumen in order to metabolize lactate produced from LAB. As
explained earlier, LAU take longer to replicate in the rumen, and
thus, animals are fed diets with increasing levels of concentrate
after being adapted to previous levels; these steps take a few
weeks to be completed. Although step-up protocols are costly,
they may eventually dilute the LPS effects from the microbial
changes, which happens either by reducing Gram-negative
bacterial lysis due to a less abrupt change of bacterial substrates
or by the diluted effects of bacterial LPS toxicity. In any way, when
step-up protocols are taken into account, besides allowing time
for LAU to adequately replicate in the rumen, these procedures
may contribute to a decrease in the LPS effects as well.
Although the literature is scarce of studies evaluating
ruminal LPS changes in animals in step-up diets, some studies
have evaluated bacterial changes during this period (Wells
et  al., 1997; Fernando et  al., 2010; Granja-Salcedo et  al., 2016).
Wells et  al. (1997) reported that microbial changes are less
expressive in animals gradually adapted to high concentrate
diets (up to 80% of cereal grain). According to the authors,
changes in ruminal pH were also not as pronounced as those
seen in animals with an abrupt transition to high concentrate
diets; thus, as ruminal pH was not a major factor for microbial
changes in their study, the observed microbial changes in the
study of Wells et al. (1997) were reported to be mostly associated
with bacteriocins produced by Lactobacillus spp.
Fernando et  al. (2010), in a similar study, allowed animals
to have 1  wk of adaptation per diet, from 100% forage to up
to 80% of concentrate in the diet. In their study, the phylum
Bacteroidetes gradually increased, while Fibrobacteres
Monteiro and Faciola  |  5
decreased. The increase in Bacteroidetes is the opposite of what
it is expected in animals transitioning to high concentrate diets,
and because Bacteroidetes is the major Gram-negative bacteria
in the rumen, further investigation is necessary. Interestingly,
two species decreased gradually in the study of Fernando et al.
(2010), Butyrivibrio fibrisolvens and Fibrobacter succinogenes. The
former is known as another major bacteriocin producers in the
rumen (Azevedo et al., 2015), while the latter is one of the main
Gram-negative cellulolytic bacteria in the rumen and contains
LPS (Ransom-Jones et al., 2012).
Also using step-up protocols, Granja-Salcedo et  al. (2016)
reported that the increase of grains in the diet (up to 80%
concentrate) was correlated with a gradual decrease in the
populations of Ruminococcus flavefaciens and Ruminococcus albus.
These are other major cellulolytic bacteria that, although they
are Gram-positive, they have been reported to produce a series
of bacteriocins in the rumen (Morrison and Miron, 2000; Brulc
et al., 2011; Azevedo et al., 2015). Therefore, the combination of
slow increases of substrate for the production of organic acids,
especially lactate, and the gradual removal of bacteria that can
contribute to ruminal acute acidosis (through bacteriocins and
LPS) may be another reason for the success of step-up diets.
However, because these protocols take weeks to be completed,
they are not usually viable economically.
Ruminal Lipopolysaccharide Toxicity
As described above, there is evidence that free LPS increases in
the rumen and hindgut of animals experiencing both SARA and
acute ruminal acidosis. There is also evidence suggesting that
Gram-negative bacterial lysis increases in the rumen ruminal
acidosis (as indicated by increased LPS concentrations), despite
some studies not finding a decrease in Gram-negative bacteria
concentration. However, we could not find many studies
that analyzed the LPS toxicity of individual ruminal bacteria,
analyzed the toxicity of the bacteria that decrease during
SARA and acute ruminal acidosis, or even that evaluated the
reason why LPS concentration in the hindgut is also elevated
in animals experiencing such problems. As mentioned earlier,
when ruminal bacterial LPS was extracted from mixed ruminal
bacteria in both hay and grain-fed animals, ruminal LPS had less
toxicity than those of E. coli and S. typhi in a variety of models
(Nagaraja et  al., 1978b). We could find only one study that, in
fact, analyzed the toxicity of a single rumen bacterium and
that was M. elsdenii (Nagaraja et al., 1979). However, the authors
concluded that M. elsdenii LPS had low potency and would likely
not be the major problem of ruminal acidosis.
Thus, three possibilities are likely: 1)  total free ruminal
LPS, although less toxic than E.  coli and S.  typhosa LPS, has
a concentration that overloads the ruminant detoxification
system and triggers the effects of ruminal acidosis; a topic
reviewed by Aschenbach et  al. (2019); 2)  free LPS from specific
bacterial groups are as toxic as that from E. coli and S. typhosa;
however, such LPS have their toxic effects diluted into the
pool of free ruminal LPS and decrease the toxicity of total free
ruminal LPS; and 3) the difference in the LPS pattern in animals
transitioning to ruminal acidosis could be a signaling pathway
by the animal to detect gastrointestinal tract (GIT) changes.
To understand which of these possibilities is more likely
to happen, it is important to understand how ruminal LPS
compares to other types of LPS, such as from E. coli. As explained
earlier, LPS is released in the GIT during bacterial lysis, growth,
and division (Petsch and Anspach, 2000; Yaron et  al., 2000);
 6 | Journal of Animal Science, 2020, Vol. 98, No. 8
however, bacterial lysis is the method that contributes the most
to free LPS concentration. The structure of LPS is composed of
three parts (Figure  1): a distal polysaccharide (O-antigen), a
core oligosaccharide, and the lipid A that attaches to the outer
membrane of Gram-negative bacteria (Raetz and Whitfield,
2002; Matsuura, 2013). The distal polysaccharide has been
reported to have up to 50 oligosaccharides composed of two to
eight sugar monomers, which may vary in a strain to species
level (Matsuura, 2013). This structure plays a major role in
bacterial colonization, adhesion, and escape from predation
(Moran, 2008). The variation of the core oligosaccharide only
happens at a higher taxonomic level, and such variation is
not as common as the variation of the distal part (Raetz and
Whitfield, 2002). These two structures have been reported to be
present in wild-type strains only and not in cultured strains
(Raetz and Whitfield, 2002). Interestingly, these structures have
been associated with bacterial resistance to antibiotics (Raetz
and Whitfield, 2002). Antibiotics have been used in ruminant
nutrition, especially in feedlot beef cattle diets in which acute
acidosis is more prevalent (Russell and Strobel, 1989). Indeed,
concerns regarding bacterial resistance by Gram-negative
bacteria to antibiotics have been raised; although these were
not associated directly with LPS at that time, the resistance was
believed to be from the outer-membrane protection of these
bacteria (Russell and Strobel, 1989; Russell and Rychlik, 2001).
Lastly, the lipid A  is the primary toxic structure of bacterial
LPS, and it is present in high concentrations in the cell-wall
of Gram-negative bacteria (around 106 residues/cell; Raetz and
Whitfield, 2002). The lipid A from LPS is the portion that attaches
to macrophage and endothelial cell receptors (Toll-like receptor
4 [TLR4]) to cause an immune response (Poltorak et  al., 1998;
Hoshino et al., 1999; Aderem and Ulevitch, 2000). However, TLR4
receptors have also been reported to be present in the surface
of human intestinal epithelial cells, and it is believed to play a
major role in the gut LPS signaling (Abreu, 2010).
Toxicity of LPS comes from the composition of lipid A (Netea
et al., 2002), as its acyl groups are correlated with TLR4 signaling,
and has been demonstrated in Salmonella typhimurium to
decrease its toxicity with decreased acylation when tested in
human macrophage cells (Matsuura et  al., 2012). These acyl
groups may vary from 4 to 6 acyl chains, being 4 and 5 acyl groups
shown to be the LPS with less toxicity (immunoinhibitory/silent
LPS), and 6 the most likely to activate receptors and cause an
immune response (E. coli LPS) (Netea et al., 2002; Matsuura, 2013).
In a study evaluating the lipid A structure of bacterial LPS from
Yersinia, Yersinia enterocolitica, and Yersinia pseudotuberculosis,
Rebeil et al. (2004) reported that acylation may vary depending
on the temperature in which these bacteria are cultured. In their
study, Rebeil et al. (2004) found that 21 °C incubation temperature
promoted hexa-acylation of lipid A  and 37  °C promoted tetra-
acylation. Interestingly, the LPS used for comparisons with the
ruminal LPS was E.  coli and S.  typhosa (Nagaraja et  al., 1978b),
which may have the most toxic LPS activities, suggesting
that in further evaluations: 1)  the lipid A  structure of ruminal
bacteria LPS should be characterized to understand its acylation
differences per bacterial group (mainly from those that decrease
during ruminal acidosis); 2)  silent ruminal LPS effects (tetra
and penta-acylated lipid A) should also be taken into account,
as silent LPS does not trigger an immune response; and 3) the
possibility of some ruminal bacteria having highly toxic lipid
A  LPS replacing those with silent LPS during ruminal acidosis
should also be considered.
Regarding LPS translocation, there are two primary
pathways in which ruminal or caecal LPS could trigger an
immune response in ruminants: paracellular and transcellular
(Tomlinson and Blikslager, 2004; Mani et  al., 2012; Eckel and
Ametaj, 2016). First, ruminal epithelial damage has been shown
to be prevalent during ruminal acidosis; because of low pH and
high concentration of organic acids, a high osmotic pressure
promotes a barrier opening and endotoxins may translocate
paracellularly into the epithelium (Aschenbach et  al., 2019).
Undigested starch reaching the large intestine in these high-
grain diets could also cause the development of lower gut
acidosis, leading to more severe epithelium damage than that
occurring in the rumen (Steele et  al., 2016), thus increasing
LPS paracellular translocation. The second pathway is the
transcellular translocation, which is mediated through TLR4
receptors explained before (Mani et  al., 2012). Of the total LPS
transcytosis by the epithelial cells from the lumen, only 15%
reaches the basolateral side, with the remaining being either
recycled back to the lumen (38%) or packed into lysosomes (47%)
(Beatty et al., 1999). However, chylomicrons have shown to have
a high affinity to LPS, and thus, LPS can also enter the Golgi
apparatus and be transported through the lymph and blood
(Ghoshal et al., 2009).
In a recent study evaluating which bacterial group could be
contributing to the pool of LPS in the human GIT, d′Hennezel
et  al. (2017) reported that the LPS from the Bacteroidales
are the most abundant in human feces (79% to 92.4% of the
total LPS). Similar to Mullenax et al. (1966) and Nagaraja et al.
(1978b), d′Hennezel et al. (2017) tested the toxicity of the total
LPS from human feces against the LPS from E.  coli, however,
in peripheral blood mononuclear cells (lymphocytes and
monocytes). The authors showed that when 10 LPS molecules
from mixed fecal samples were present in the media with 1
E.  coli LPS, TLR4 signaling was not activated, which in some
cases only 1 LPS from mixed fecal samples was able to stop
the activity of 1 LPS from E.  coli. Later, in order to evaluate if
the LPS immunoinhibitory capacity could be an order-wide
feature, the authors tested the toxicity of LPS only from species
of the order Bacteroidales against E.  coli LPS in similar cells.
d′Hennezel et  al. (2017) showed that from the Bacteroidales
LPS species tested, all were underacylated, which caused an
inhibitory effect on TLR4 signaling (silent LPS), thus showing
that immunoinhibitory features of LPS may be conserved at
higher taxonomic levels.
Furthermore, d′Hennezel et  al. (2017) extracted the
extracellular polymeric matrix (EPM; or extracellular polymeric
substances) from 29 strains of the 4 main phyla in the human GIT
in order to evaluate its association with inflammation. The EPM
is a matrix with active compounds surrounding the bacterial
cell wall that helps in the colonization process through biofilm
formation and intercellular aggregation (Rossi et  al., 2015).
The authors reported that the bacterial EPM downregulated
inflammation through TLR4 and TLR2 by 4- to 20-fold in primary
human immune cells, with the EPM of Bacteroidales reducing
inflammation the most; yet, EPM from Bacteroidales had one of
the highest LPS contents of the bacterial EPM tested. This study
was performed with fecal samples, which contain a less diverse
microbiome compared with rumen, thus providing a variety
of LPS inferior to that of the rumen. However, these findings
from d′Hennezel et al. (2017) can potentially bring new context
to ruminal acidosis research, as Bacteroidales comprise some
of the main genera found in the rumen, such as the Prevotella
spp. as explained previously (Henderson et al., 2015). Also, their
research sheds light on how future studies could analyze the
acylation of specific ruminal bacteria and test their toxicity in
the models herein tested.

Lastly, Mani et  al. (2013) using pigs as a model to
study intestinal endotoxin transport reported that n-3
polyunsaturated fatty acids (PUFA) decreased LPS signaling
possibly by also modulating TLR4, and that saturated fatty acid
(SFA) supplementation increased LPS serum concentration.
The mechanism by which fatty acids modulate TLR4 receptor is
directly associated with the lipid A composition of LPS. Nontoxic
LPS has been reported to have PUFA in its lipid A composition
and showed an antagonistic effect in mice when compared
with LPS with lipid A  containing only SFA in its composition
(Qureshi et  al., 1991). On the other hand, SFA (e.g., lauric and
myristic acids) are part of the lipid A  of LPS, and thus, SFA in
the lumen induce TLR4 activation, triggering LPS absorption (Lee
et  al., 2004). Although cattle diets have low fatty acid content
(<7% ether extract), the majority of the fatty acids that leave the
rumen are SFA, which in high LPS conditions, such as in SARA
and acute acidosis, this could be another possibility of triggering
LPS absorption and an immune response.
Conclusion Remarks
In conclusion, although pH and lactate have been used as
parameters for the pathogenesis for ruminal acidosis, there is
evidence that LPS could greatly contribute to acidosis. The studies
described here indicate that ruminal acidosis causes a dysbiosis
in the ruminal microbiome, and some Gram-negative bacteria
decrease their abundance in the rumen and lysis of these bacteria
increase, as indicated by the increase in free LPS. Greater LPS
concentration during ruminal acidosis has also been reported in
the hindgut, which increases the chances of evoking an immune
response. Other studies show that besides LPS increasing in the
rumen during this time, increased LPS could also trigger the
growth of ruminal bacteria associated with acidosis. Therefore,
future studies should be performed to characterize ruminal
LPS and its lipid A structure in order to accurately determine its
toxicity so that more strategies could be developed in order to
overcome this major challenge in ruminant nutrition.
Conflict of interest statement
The authors declare no real or perceived conflicts of interest.
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