Endotoxins in Cows an Under Estimated Risk?

Endotoxins in cows An under estimated risk?

Simone Schaumberger Product Manager, Mycotoxin Risk Management

Nicole Reisinger Project Leader, Endotoxins Endotoxins in cows An under estimated risk? Simone Schaumberger Product Manager, Mycotoxin Risk Management Nicole Reisinger Project Leader, Endotoxins

An under estimated risk? Endotoxins are incredibly fascinating substances. On the one hand, they stimulate the immune system in a positive way, yet on the other hand cause endotoxic shock and death.

oxins are known to have rumen permeability, allowing endotoxins negative eff ects on rumen to enter the organism. But what does this fermentation. In general, mean for your cow? two types of toxins have drawn much attention About endotoxins within animal health and welfare: toxins Endotoxins have been known since Endotoxins are… Tfrom fungi (mycotoxins) and bacterial tox- the early 1900s because of their pyro- • Produced from gram- ins (endotoxins, exotoxins). genic (fever inducing) eff ect. In general, negative bacteria Th ere is growing attention on the is- endotoxins are parts of the cell wall of all • Part of the bacterial cell wall sue of increased endotoxin values in the gram-negative bacteria (Figure 1) and they • Macromolecules with 300,000 rumen during rumen acidiosis. High are of great interest because of their eff ect to 1,000,000 dalton carbohydrate diets change the microfl o- on the immune system. Endotoxins are • Pyrogens (can induce fever) ra in the rumen, leading to the death of also called lipopolysaccharides (LPS) as gram-negative bacteria and an increase in their structure consists of a lipid (lipid A, • Abundant in the rumen and gram-positive bacteria. Th is eff ect leads to immunogenic part, lowest variability) and gastrointestinal tract a dysbiosis, which in turn results in rum- a polysaccharide (species specifi c part, high • Present in the air, water and feed • Heat and pH stable

initis. Ruminitis consequently increases variability of chain length). Photo: Eraxion_iStockphoto

Figure 1. Comparison of gram-positive and gram-negative bacterial cell wall. The location of LPS in the cell wall is circled.

Gram-positive Gram-negative bacteria bacteria

Plasma membrane Outer membrane

Lipoproteins

DNA Oligomers Core glycolipid O-specific polysaccharide chain Endotoxin (Lipopoly- saccharide) lipid A n Superantigens O-specific (outer) (inner) (Protein) oligosaccharide core oligosaccharide subunit

Bacterial death Exotoxins (Antibiotics, temperature) (Protein)

Lipoteichoic (Murein) Acid Peptidoglycan Source: BIOMIN Simone Schaumberger Product Manager, Mycotoxin Risk Management Nicole Reisinger Project Leader, Endotoxins

Risk factors for endotoxin-related diseases in ruminants

Table 1. Summary of endotoxin activity (endotoxin units, EU/ml) in differ- tion of special kinds of antibiotics (for ex- ent parts of the cow in healthy animals and animals with experimentally ample beta-lactam antibiotics) with bacte- induced sub-acute ruminal acidosis (SARA) from different studies. ricidal activity may increase the liberation EU/ml EU/ml of endotoxins. This fact should be taken Healthy cow SARA into consideration when treating a cow Blood < 0.05 EU/ml 0.05 – 1 EU/ml with antibiotics. Rumen 3,700 – 30,000 EU/ml 120,000 – 210,000 EU/ml Ileum 4,000 EU/ml 110,000 EU/ml Effects in ruminants Cecal 18,000 EU/ml 130,000 EU/ml Ruminants are constantly in contact with endotoxins via feed, air and the en- Fecal 14,000 EU/ml 100,000 EU/ml vironment. In healthy animals, only small Source: Adapted from Plaizier et al., 2013 quantities are absorbed into the blood through the intestine. They are then trans- The structure of the LPS is crucial for ported and detoxified in the liver. Due the uptake and detoxification of the mol- to their structure, endotoxins can also be ecule. Endotoxins are released during the stored in the fat tissue. death or overwhelming proliferation of In healthy ruminants, endotoxins are gram-negative bacteria. The administra- present at certain concentrations in the rumen, intestinal tract and feces. In the case of energy deficiencies or feed imbalances, 1 Rumen simulation model the rumen or gut wall becomes more per- The rumen simulation is an important in vitro model to test the influence of feed meable, which allows more endotoxins to additives on rumen physiology. The model was adapted at the Biomin Research Center (Re- enter the bloodstream. If the animal lacks search Team Analytics) to determine, with the use of natural rumen fluid, the influence of sufficient energy, fat is degraded and even additives on the rumen pH, bacterial number and concentration of fatty acids in a reactor (pictured below). The influence on endotoxin concentration in the rumen can also be tested. more endotoxins can enter the organism. Endotoxin concentrations can increase and can be measured in the blood (Table 1). This may trigger a range of diseases such as mastitis, endometritis, laminitis, dermatitis digitalis and endotoxic shock, among others.

In vivo meets in vitro Endotoxins are receptor-mediated agents and hence, their predictive value in animals is uncertain, especially via the oral route. Inducing a controlled in vivo oral endotoxin challenge via feed is a difficult task. Therefore, in vitro experiments provide an opportunity to explain the mechanism Endotoxins in cows An underestimated risk?

Cows in early lactation Primiparous cows Cows grazing or fed with rapidly fermentable low fi ber grass High amount of concentrates Sub-acute acidosis Management in stables

Figure 2. Comparison of mean endotoxin Figure 3. Signiﬁ cant decrease in separation values of reactors from the rumen simula- force [%] from explants treated with 10 and 2 Ex vivo tion model. The antibiotic treated reactors 100 µgl/ml LPS compared to negative control laminitis model (green) showed signiﬁ cantly higher endo- (green). toxin values after long-term incubation. (A) Dissection of the claw

25,000 ** 100

20,000 80 ] EU/ml ]

% * 15,000 60

10,000 40 * (B) Cultivation of explants

5,000 20 Separation force [ Endotoxin activity [ 0 0 Untreated Antibiotic Control LPS 1 LPS 10 LPS 100 reactors treated mg/L mg/L mg/L reactors Source: BIOMIN, 2014 Source: BIOMIN, 2014 Explants of connective tissue, lamellae and claw wall. • 24 hours induced by endotoxins. Th e rumen simu- required to separate the connective tissue • 37°C, 5% CO2 lation model provides a method to test the from the lamellae (Figure 3). • 0 – 100 µg/ml LPS eff ects of feed additives (Box 1). Preliminary results with the rumen Conclusion (C) Testing of separation force simulation model confi rmed that antibi- Th e damages caused by endotoxins are otics have a negative eff ect on endotoxin fact and no fi ction. Th ey are ubiquitous production in the rumen. After a two- in the environment and are permanently week long incubation, the endotoxin con- released. A healthy cow can cope with the centration of the reactors treated with an- normal load of endotoxins by detoxifi ca- tibiotics increased signifi cantly compared tion in the liver or the lymph. to the untreated reactors (Figure 2). Th is When there is an increase in endo- shows the need for alternative strategies toxins or liver failure, endotoxins can to positively infl uence the rumen physi- overwhelm the cow’s biological function. ology and control the endotoxin load in Infl ammation cascades are triggered and the rumen. result in diff erent diseases, which, in the Another in vitro model is the ex vivo worst cases, may lead to shock and death. laminitis mo del (Box 2), which uses claw As endotoxins are ever present in the tissue to test the eff ects of endotoxins. Th is ruminant environment, control strat- model demonstrates that endotoxins have egies to prevent endotoxin-related dis- a negative eff ect on the claw tissue. En- eases among cows are essential, and re- dotoxins signifi cantly decreased the force commended.

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