Endotoxin and animal health; A hidden threat in animal feed

Endotoxin can have detrimental effects on susceptible animals leading to an increase in illness, death rate and economic losses. Regular testing & monitoring of endotoxin levels in raw ingredients and finished feed can enhance animal welfare & improve livestock productivity. Developing endotoxin-specific binders would reduce the burden on animal health & provide financial benefits for the grower & farmer.

Dr Anas Sattar
Co-founder & Senior Research and Innovation Scientist
Molendotech Ltd

Endotoxin is a complex molecule consisting of lipid and polysaccharide moieties present on the outer membrane of Gram-negative bacteria. Endotoxin consists of 3 regions: O specific chain, core region and lipid A as shown in figure 1.

Endotoxins are ubiquitously found in the environment, and their potency mainly depends on the molecular structure of Lipid A. Typically, lipid A consists of 6 acylated chains and two phosphate groups, as shown in figure 2. The changes in the chemical structure of Escherichia coli’s lipid A (represented by red circles for phosphate and a red rectangle for the acyl group) and the approximate fold decrease in biological activity are also illustrated.

Figure 1. Structure of endotoxin. Adapted and modified from Alexander and Rietschel (2001), cited in Sattar (2014).
Figure 2. Chemical structure of lipid A. of (A) Escherichia coli and (B) Bacteroides fragilis. Adapted and modified from (Weintraub et al., 1989) and (Rietschel et al., 1993).

Certain Gram-negative bacteria (such as Enterobacteriaceae, Bacteroidetes, and others) are present as normal flora in the intestinal tract of humans and animals. Endotoxin is constantly shed from Gram-negative bacteria during multiplication through binary fission or when they die. Other potential sources of endotoxin contamination include water, feed, certain feed additives produced by E. coli (including vitamins and amino acids), faeces from farm animals, and the environment.

Endotoxins can translocate from the animal gut to the systemic circulation when the tight junctions between gut epithelial cells become compromised. This can occur due to dietary components, injury or stress. Additionally, endotoxins can translocate to the bloodstream from the mammary gland (Dosogne et al., 2002) and uterus (Mateus et al., 2003, cited in Eckel and Ametaj, 2020).

The mechanism of endotoxin recognition starts when LPS-binding protein (LBP) binds to endotoxin, which then binds to CD14 molecules on the cell surface of immune cells. LBP-endotoxin-CD14 complex binds to the extracellular domain of TLR4-MD2, a receptor for LPS predominantly expressed on immune cells such as macrophages and dendritic cells. This binding will cause TLR4 to activate a signalling cascade resulting in the production of mediators that produce the inflammatory response and recruit other immune cells to the site of infection. (Poltorak et al., 1998, O’Neill and Dinarello, 2000, Keating et al., 2007, Sun et al., 2004, Kim et al., 2007).

Certain Gram-negative bacteria can be pathogenic to animals and can lead to the inflammation of the uterus wall (Metritis) and inflammation of mammary glands and udder tissue (Mastitis). Endotoxins can also cause various health disorders, including fatty liver and ruminal acidosis. These conditions can result in a reduction in feed intake and subsequently decrease weight gain.

In weaning piglets, endotoxin (lipid A) has numerous effects associated with infection. These effects include fever, changes in blood cell population, activation of macrophages, adjuvanticity (enhancement of immune response), and induction of various inflammatory mediators. Endotoxins are also implicated in the pathogenesis of oedema disease and endotoxin shock (van Beers-Schreurs et al., 1992). These conditions are characterized by severe inflammatory responses and can have detrimental effects on the health and well-being of piglets.

In a study conducted by Parra and colleagues (2011), the effect of endotoxin on the intestinal villi of swine was investigated. The study concluded that endotoxin had a negative impact on the morphology of the intestinal villi. Specifically, it resulted in a reduction in both the height and the area of the villi. Additionally, endotoxin caused an increase in the width and depth of the intestinal glands.

The authors of the study suggest that these changes in the morphology of the intestinal villi and glands are responsible for a decrease in nutrient absorption and an increase in susceptibility to infections. Ultimately, these effects can contribute to the development of post-weaning diarrhoea syndrome.

Indeed, increasing disease and subsequent deaths in livestock can result in significant economic losses. These losses can arise from various factors, such as decreased productivity, increased veterinary and medical expenses, loss of market value, and potential disruptions in supply chains.

Studies conducted by Wallace et al. (2016) and Eckel and Ametaj (2020) have highlighted the economic impact of livestock diseases. These impacts can extend beyond the immediate costs of treatment and mortality, affecting the profitability and sustainability of the livestock industry. Therefore, it is crucial to implement effective disease prevention and control measures to mitigate economic losses and promote the well-being of livestock populations.

According to Huntley et al. (2017), stimulating tested animals with endotoxin increased total heat production by 19%, increased maintenance energy requirements by 23%, and decreased lipid deposition by 27% and average daily gain by 26%.

According to DEFRA, there are approximately 5 million pigs in the UK and 150 million in the EU, with average weight of 80 kgs. Feeding represents 65% of the cost of the pig (£1.06/kg). A 15% improvement (endotoxin studies) would mean a gain of:

5(M) X 80 (kgs) X 1.06 (£/kgs) x15%=£63M per annum

Similarly, the calculation for each farm can be estimated using the following formula:

(Number of animals) x (average weight of animals) x (Cost of feed) X 15% = £gain

Livestock biosecurity is defined as the prevention of microbes from causing a risk to farm animals, farm workers, the safety and quality of a food product and the environment.

Mitigation strategies must be implemented to minimise and control Gram-negative bacteria and endotoxin. These mitigations can be divided into two categories: outside and inside the animal:

1. Outside the animal:
Regular endotoxin testing of raw cereal, finished animal feed products, storage facilities, water and water storage/pipes, environment and litter. Various raw ingredients such as maize, barley, wheat, oats, triticale, beans, soya and other cereals have different levels of endotoxin based on the source of where they come from and storage conditions. Therefore, testing the above raw ingredients can guarantee that the animals are not exposed to high endotoxin levels; the same applies to finished products.

2. Inside the animal:
Pre- and pro-biotics and feed additives would help reduce and control bacterial and endotoxin levels.

Developing binders that specifically bind to endotoxin and including them in animal feed will reduce the availability of endotoxins inside the animal and reduce the detrimental effects of endotoxin on animal health.

Endotoxin levels present in animal feed components are typically set by regulatory bodies, standards established by the industry, European Union (EU) and European Feed Additives and Premixtures Association (FEFANA), and recommendations from the scientific community. These guidelines aim to prevent adverse effects associated with high levels of endotoxin, such as inflammation, immune responses, and negative impacts on animal health. The acceptable or permissible levels of endotoxin in animal feed can differ depending on the species of animals being targeted and the particular feed component in question. Table 1 summarises the maximum allowed level of endotoxin:

It is important to acknowledge that endotoxin sensitivity varies among different animal species, and certain animals, such as pigs and poultry, are commonly more susceptible and hence more affected by endotoxin.

Endotoxin is a complex molecule on the outer membrane of Gram-negative bacteria that affects susceptible animals, leading to increased illness and death and subsequent economic losses.

To improve livestock productivity, reduce the risks associated with endotoxin-related diseases and increase financial benefits to the grower, it is recommended to:
A- Regularly monitor endotoxin levels in animal feed
B- Develop endotoxin binders and include them in the feed
C- Adhere to best practices established by the industry and scientific community
D- Seek guidance from experts to provide specific recommendations for minimizing these risks

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