ENDOTOXINS in FEED and their relation to MYCOTOXINS
Prof. Simon Jackson Molendotech Ltd., Devon, UK
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What are endotoxins? Endotoxins are major components of the outer membrane of Gram-negative bacteria. They are composed of a lipid anchor, termed lipid A, and a polysaccharide repeating unit. Due to the structure, endotoxins are also known as lipopolysaccharides (LPS) (Figure 1).
Figure 1. Schematic structure of endotoxin from E. coli. (Adapted from Abate et al., Journal of Medical Microbiology, 2017).
Endotoxins increase the negative charge of the cell membrane and help to stabilize the overall membrane structure forming an effective permeability barrier against small, hydrophobic molecules making Gram-negative bacteria innately resistant to many antimicrobial compounds.
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While the overall structure of endotoxin is similar
In addition to their role in bacterial
among Gram-negative bacterial species, changes
stability and identification, endotoxins
in the polysaccharide composition (O-specific
are highly implicated in human and
chain or O-antigen) generate great variety,
animal disease, particularly in relation to
making it different for different bacterial strains.
immune and inflammatory responses.
In fact, the O-antigen is used to identify specific strains of enteric bacteria such as E. coli (e.g., E. coli O157), where the ‘O’ stands for O-antigen.
What is the biological activity of endotoxins? The biological activity of endotoxins is
It is important to remember that endotoxins are
associated with both the lipid and polysaccharide
particularly heat-stable and are not destroyed by
components of lipopolysaccharide (LPS).
normal autoclaving or heat-inactivating methods. Moreover, endotoxins, unlike bacteria, are not
Toxicity is associated with Lipid A. Immunogenicity is associated with
affected by antibiotics and may produce their toxic effects in the absence of viable bacteria.
the polysaccharide components. Lipid A is a powerful biological response modifier that can stimulate the mammalian immune system. It anchors the endotoxin molecule in the outer membrane of bacterial cells, exerting its biological effects when the endotoxin is released from bacterial cells either due to natural shedding from growing bacteria or when the bacteria are lysed by autolysis, complement attack, phagocytosis, or by certain antibiotics.
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What is the difference between endotoxin and exotoxin? Endotoxins, per se, are not toxic like other toxins such as exotoxins. Exotoxins are proteins produced and secreted by some strains of Gram-positive and Gram-negative bacteria and they
In contrast, endotoxins are released upon bacterial
usually have specific target cells where
lysis and do not have specific target cells.
disrupting metabolism and cell death.
For example, enterotoxigenic E. coli (ETEC) produce a heat-stable enterotoxin (STa) that binds to receptors on enterocytes in the gut, disrupting ion and water transportation which leads to abundant diarrhoea (Kopic and Geibel, 2010).
ENDOTOXINS
EXOTOXINS
they cause toxic effects, including
They are ‘sensed’ by the immune system as a signal of infection that triggers inflammatory immune responses that can result in life-threatening conditions (sepsis).
How do endotoxins stimulate the immune system? Endotoxin-associated ‘toxicity’ is due to the
Immune cells, such as macrophages
host’s immune response. Endotoxins are
and monocytes, have cell surface
recognized by the immune system as signals of
receptors -Toll-like receptor 4 (TLR4)-
a Gram-negative bacterial infection, triggering
that recognize endotoxins.
an innate inflammatory immune response. Binding of the endotoxin to TLR4 triggers a In the blood, which is normally sterile, minute
cell signalling cascade that results in gene
quantities of endotoxins (<1ug) can stimulate
expression and the production of proteins
an intense inflammatory response.
that stimulate an inflammatory response:
Cytokines: Interleukin 6 (IL-6) and TNFα. Inflammatory mediators: Platelet Activating Factor (PAF) and Interleukin 1 (IL-1).
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Endotoxins also activate the Complement system
ANIMAL FEED
and trigger the blood clotting system (Figure 2).
IMMUNE CELLS Figure 2. Endotoxins present in animal feed can activate the host’s immune system, causing an inflammatory response that may damage tissues and organs and lead to death.
Inflammatory gene expresión: TNFα, IL-6, IL-1 Lipid mediators (PAF)
ENDOTOXIN
ACTIVATED COMPLEMENT
BLOOD CLOTTING
INFLAMMATION
This stimulates further inflammatory
The liver also serves an
responses, allowing a rapid activation of
important role in this regard.
the innate immune system to deal with any bacteria before the infection develops too far. However, the powerful inflammatory response has to be carefully controlled and ‘switched off’ to avoid damage to the host. This is achieved by the production of anti-inflammatory mediators such as Interleukin 10 and Interleukin 1 receptor
If a significant amount of endotoxins enters the bloodstream or cannot be removed quickly, the inflammatory response may become exaggerated with a ‘cytokine storm’ that escapes control of anti-inflammatory mechanisms, damaging the host’s tissues and organs.
antagonist and proteins that bind endotoxins (LPS binding protein (LBP).
This condition is known as sepsis and carries a high mortality rate.
Moreover, Immune cells such as macrophages and neutrophils can
The presence of endotoxins in the
help detoxify endotoxins through
blood can therefore be lethal and
production of enzymes that degrade
mechanisms that allow its access to
the Lipid A part of the molecule.
the circulation should be minimized.
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How are endotoxins detected? Response to endotoxins has been conserved
FACTOR C
through evolution and therefore endowed our ancestors with survival advantage in a world full of Gram-negative pathogens.
ENDOTOXIN
Indeed, a response to endotoxins has been
ACTIVATED FACTOR C
seen in ancient marine creatures such as the horseshoe crab (Limulus polyphemus) that have been on earth for many millions of years (Levin and Bang, 1968). The discovery of a blood clotting immune response to endotoxins in the horseshoe crab
CLOTTING CASCADE ENZYMES
led to the development of a very sensitive assay for bacterial endotoxins using the blood cells (amebocytes) from these organisms - the Limulus amebocyte lysate (LAL) assay (Figura 3).
COAGULIN Figure 3. Principle of the LAL assay to detect bacterial endotoxins. Endotoxins, shed from Gram-negative bacteria, bind and activate Factor C in the Limulus amebocytes. This triggers a cascade reaction of enzymes that culminates in the formation of a blood clot (coagulin). Addition of a chromogenic substrate allows a coloured product to be produced by the reaction.
CHROMOGENIC SUBSTRATE
COLOURED PRODUCT
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This assay is routinely used in the pharmaceutical industry as all drugs and medical devices such as pace makers, catheters and other invasive devices are required to be free from endotoxin contamination as potentially life-threatening immune reactions (sepsis) could be elicited if endotoxin is administered inadvertently to the human body (Abate et al.,2020).
Researchers at Molendotech Limited have developed a version of the LAL assay that can test environmental samples for endotoxin content as a screen for bacterial or fecal contamination of water or foodstuffs including animal feed.
How can endotoxins enter the circulation? The gut is a huge reservoir of Gram-negative
Compromise of gut barrier function is also
bacteria and, consequently, contains large
a concern in animals and stress in pigs or
quantities of endotoxins that can enter the
sub-acute ruminal acidosis (SARA) in cattle
bloodstream through intestinal absorption.
are known to facilitate endotoxin entry to the circulation and drive pathological consequences.
The body has developed compartmentalization to prevent high amounts of endotoxins from entering the bloodstream. However, gut lesions, a lipidrich diet, drug treatments, infections, or immature status (e.g., neonates) facilitate the translocation of endotoxins across the membrane into the systemic circulation.
Low doses of endotoxins have been shown to induce the symptoms of SARA, including inflammation, lowering ruminal pH and altered microbiota (Jing et al., 2014). Therefore, Inhalation or consumption of endotoxins can initiate a serious disease in young animals. Feed additives, such as amino acids and vitamins are increasingly being produced by recombinant technology using Gram-negative bacteria, especially E. coli.
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Therefore, it might be worthwhile to limit
If endotoxins from the bacteria
endotoxin-containing feed ingredients in
contaminate the additive, it
animals with a disturbed gastrointestinal
could pose a risk to animals,
barrier function (Wallace et al., 2016).
as well as workers handling the additives and the consumer.
It would also be expected that animals might react to inhaled endotoxins present in dust in the sheds and from the feed, in a similar manner to human workers. However, there are few studies
It has been documented, for example, that workers exposed to endotoxins inhaled with
on animal exposure to inhaled endotoxins and the limits of exposure are uncertain
dust from poultry sheds or other sources exhibit clinical symptoms, including decreased lung function (Health Council of the
Netherlands, 2010).
Although farm livestock is continuously exposed to endotoxins from the environment, including feed, diets containing high concentrations of endotoxins pose a risk as even small doses crossing the gut barrier can cause serious disease (Mani et al., 2013).
Can MYCOTOXINS influence the response to endotoxins? Mycotoxins are toxins produced by fungi (molds) growing on crops or on feed ingredients during storage. They can cause a variety of adverse health effects and pose a serious health threat to both humans and livestock. Mycotoxins commonly found in feed include aflatoxins, zearalenone, T2-toxin, deoxynivalenol, and ochratoxin A.
Contaminated feed will result in the presence of both mycotoxins and endotoxins together in the gastrointestinal tract of production animals and it has been found they have synergistic adverse effects on animal health and consequent economic impact.
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As noted earlier, an intact gut barrier is important
Mycotoxins also have effects on the immune
to reduce the uptake of endotoxins into the
cell function and damage the liver, resulting in
circulation and to prevent their inflammatory
increased inflammatory responses while decreasing
consequences. Moreover, a healthy immune
any endotoxin removal mechanisms (Figure 4).
system and liver are also important to control the amount of endotoxin in the blood. Mycotoxins damage the gut epithelia (barrier) thus allowing more gut contents, including endotoxins, to transmigrate to the circulation.
Thus, mycotoxins present in feed will act synergistically with endotoxins to produce severe health consequences for animals that ingest the contaminated feed. Young, weaning, animals will be particularly vulnerable (Wallace et al., 2016).
Figure 4. Mycotoxins affect the immune cells and the liver, preventing endotoxin-detoxification enzymes and other anti-inflammatory molecules being produced. They can also disrupt the gut epithelial barrier that normally prevents endotoxin entry to the circulation.
Immune cells
Detoxification mechanisms
Liver
Endotoxin
Gut epithelium barrier
Mycotoxin Endotoxin
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How can we mitigate the effects of endotoxins and mycotoxins in feed?
To limit or prevent the effects of endotoxins and mycotoxins in feed, the feed should be treated with agents to bind and remove these molecules (Boyacioglu, 2019) and their efficiency to remove endotoxins should be tested.
Tests for different mycotoxins exist but are more complex. General screening for
In addition, the feed itself can be tested for
fungal (mold) contamination should be
Gram-negative bacterial and endotoxin content.
possible with tests being available soon.
REFERENCES
Abate W, Sattar A, Liu J, Conway ME and Jackson SK; J Med Microbiol. 2017 66(7):888-897. Abate W, Alrammah H, Kiernan M, Tonks AJ, Jackson SK Sci Rep. 2020 Jun 25;10(1):10355. Boyacioglu H. World Grain ; 2019-12-23 Health Council of the Netherlands. Endotoxins 2010. The Hague: Health Council of the Netherlands; 2010. publication no. 2010/04OSH Kopic S. and Geibel J.; Toxins 2010; (8): 2132–2157. Levin J, Bang FB.; Thromb Diath Haemorrh. 1968 19(1):186-97. Mani V,et al., : J Anim Sci. 2013; 91:2141–50. Jing L, Zhang R, Liu Y, Zhu W, Mao S. Br J Nutr. 2014;112:170–82. Wallace RJ et al., Environmental Health (2016) 15:5
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