Endotoxins in feed and their relation to mycotoxins

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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