How lipopolysaccharides cause disease

LPS are rather large and structured chemical molecules with a weight of over 100,000 D. They are highly thermostable; boiling in water at 100°C for 30 minutes does not destabilize their structure. LPS consist of three chemically distinct sections: a) the innermost part, lipid A, consisting mostly of fatty acids; b) the core, which contains an oligosaccharide; and c) the outer section, a chain of polysaccharides called O-antigen (Figure 1).

Figure 1: Structure of an LPS

The toxicity of LPS is mainly caused by lipid A; however, both lipid A and O-antigen stimulate the immune system. This happens when the LPS pass the mucosa and enter the bloodstream or when they attack the leukocytes.

The intestinal mucosa is the physical immune barrier that protects the microvilli from external agents (bacteria, free LPS viruses, etc.). Despite its strength (the thickness, for example, amounts to ≈830 µm in the colon and ≈123 µm in the jejunum), vulnerable points exist (cf. Zachary 2017).

LPS can easily come into contact with the cells of the lamina propria (a layer of connective tissue underneath the epithelium) through the microfold (M) cells of the Peyer’s patches (which consist of gut-associated lymphoid tissue). The M cells are not covered by mucus and thus exposed.

Secondly, LPS can also pass through the mucosa, where they become entangled in this gelatinous structure. There, they come into contact with the lymphocytes or can reach the regional lymph nodes through the afferent lymphatic vessels.

Thirdly, LPS might affect the tight junctions, the multiprotein complexes that keep the enterocytes (cells that form the intestinal villi) cohesive. By destabilizing the protein structures and triggering enzymatic reactions that chemically degrade them, LPS can break the tight junctions, reaching the first capillaries and, consequently, the bloodstream.

The presence of endotoxins in the blood, endotoxemia, can trigger problematic immune responses in animals. An innate immune stimulation leads to an increase in the concentration of pro-inflammatory cytokines in the blood and, consequently, to an induced febrile response in the animal: heat production increases, while the available metabolic energy decreases.  As a result, performance suffers, and in the worst-case scenario, septic shock sets in. Furthermore, when LPS compromise intestinal integrity, the risk of secondary infections increases, and production performance may decline.

LPS’ modes of action

How does all of this happen? The physiological consequences of endotoxemia are quite complex. Simplified, the immune system response to LPS in the blood takes three forms:

• The stimulation of TLR4 (toll-like receptor 4) induces monocytes and macrophages to secrete critical pro-inflammatory cytokines, primarily interleukin (IL) IL-1β, IL-6, IL-8, and tumor necrotic factor (TNF) α and β. TLR4 is a structure on the cell membrane of mainly macrophages and leukocytes, which is activated by the LPS-binding protein (LBP).
• The complement cascade constitutes about 10% of plasma proteins and determines the chemotaxis and activation of leukocytes. It can form a membrane attack complex (MAC), which perforates the membranes of pathogenic cells, enabling lysis.
• The Hagemann factor, also known as coagulation factor XII: once stimulated by LPS, it initiates the formation of fibrin (through the intrinsic coagulation pathway), which might lead to thrombosis. The Hagemann factor directly stimulates the transformation of prekallikrein to kallikrein (enzymes involved in regulating blood pressure).

Figure 2: How LPS leads to endotoxemia – 3 modes of action

These three modes of action of inflammatory stimulation lead to important physiological reactions:

• Pro-inflammatory cytokines (see above) modulate the functional expression of other immune cell types during the inflammatory response;
• Metabolites of arachidonic acid (prostaglandins, leukotrienes, and lipoxins), intra- and extracellular messengers that influence the coagulation cascade;
• Synthesis in the blood of bradykinin, a peptide responsible for the typical symptoms of inflammation, such as swelling, redness, heat and pain;
• PAF (platelet-activating factor), which creates inflammatory effects through narrowing of the blood vessels and constriction of the airways, but also through the degranulation of leukocytes.

The symptoms of endotoxemia are:  hypotension, metabolic acidosis, hemoconcentration, intestinal hemorrhage, fever, activations of neutrophils and endothelial cells, and predisposition to thrombosis.

In case of a progression to septic shock, the following sequence takes place:

1) Reduction in blood pressure and increased heart rate (hemodynamic alterations)

2) Abnormalities in body temperature

3) Progressive hypoperfusion at the level of the microvascular system

4) Hypoxic damage to susceptible cells

Up to here, symptoms follow a (severe) endotoxemia pathogenesis. A septic shock furthermore entails:

5) Quantitative changes in blood levels of leukocytes and platelets

6) Disseminated intravascular coagulation (see Hageman factor)

7) Multi-organ failure

8) Death of animal

If an animal is continously challenged with endotoxins, experiences septic shock, or comes close to it, it risks developing LPS tolerance, also known as CARS (compensatory anti-inflammatory response syndrome). This syndrome essentially depresses the immune system to control its activity. The anti-inflammatory prerogative of CARS is not to interfere directly with the elimination of pathogens but to regulate the “excessive” inflammatory reaction in a hemostatic way. However, this regulation can be extremely dangerous as the syndrome involves a lack of homeostasis control, and an excessive depression of the immune system leaves the organism exposed to the actual pathogens.

Farm animal research on endotoxemia pathogenesis

Lipopolysaccharides are difficult to quantify in the intestine of a live animal. One way to evaluate a possible endotoxemia is to analyze biomarkers present in the bloodstream. The most important one is the LPS themselves, which can be detected in a blood sample taken from the animal via ELISA. Other biomarkers include pro-inflammatory interleukins, such as TNF α and β, IL-6 or IL-8, and fibrin and fibrinogen (though they are not specific to endotoxemia). It is vital to carry out a blood sample analysis to deduce a possible endotoxemia from symptoms and performance losses in the animal.

How the metabolic effects of endotoxemia depress performance

One of the biggest issues caused by endotoxemia is that animals reduce their feed intake and show a poor feed conversion rate (FCR). Why does this happen? The productive performance of farm animals (producing milk, eggs, or meat) requires energy. An animal also requires a certain baseline amount of energy for maintenance, that is, for all activities related to its survival. As a result of inflammation and all those physiological reactions mentioned above, endotoxemia leads to a feverish state. Maintenance needs to continue; hence, the energy required for producing heat will be diverted from the energy usually spent on producing milk, egg, meat, etc., and performance suffers.

The inflammation response can result in mitochondrial injury to the intestinal cells, which alter the cellular energy metabolism. This is reflected in changes to the levels in adenosine triphosphate (ATP), the energy “currency” of living cells. A study by Li et al. (2015) observed a respective reduction of 15% and 55% in the ATP levels of the jejunum and ileum of LPS-challenged broilers, compared to the unchallenged control group. This illustrates the extent to which animals lose energy while they experience (more or less severe) endotoxemia.

Figure 3: Reduction in ATP level in Jejunum and Ileum in broilers (adapted from Li et al., 2015)

A piglet study by Huntley, Nyachoti, and Patience (2017) took this idea further (Figure 4):  3 groups of 10 Yorkshire x Landrace pigs, weighing between 11 and 25 kg, were studied in metabolic cages and in respiratory chambers. This methodology allows for simultaneous measurement of oxygen consumption, CO₂ production, energy expenditure, physical activity, and feed/water intake. The study found that LPS-challenged pigs retained 15% less of the available metabolizable energy and showed 25% less nutrient deposition. These results show concrete metabolic consequences caused by the febrile response to endotoxemia we discussed above.

Figure 4: Retained Energy as % of ME intake and nutrient deposition of pigs in metabolic cages (adapted from Huntley, Nyachoti, and Patience, 2017)

Control treatment (CON) = Pigs fed by a basal diet
Immune system stimulation treatment (ISS) = Pigs given LPS (E. coli serotype 055:B5) injection

A loss of energy retained due to a reduction in available metabolizable energy leads to losses in performance as the amount of energy available for muscle production and fat storage will be lower. Furthermore, the decrease in feed intake creates a further energy deficit concerning production needs.

A trial carried out at the University of Illinois examined the effects of repeated injections of 400 μg E. coli LPS on chick performance from 11 to 22 days after hatching. The chicks were fed casein-based diets with graded levels of arginine. LPS administration reduced weight gain (P<0.05) and feed intake, and these effects tended to be worse at higher levels of arginine supplementation (Figure 5). The researchers hypothesize that, in response to endotoxin and elevated cytokine levels, macrophages use more arginine to produce nitric oxide, diverting it from protein production for muscle development.

Figure 5: Effects of LPS on feed intake and body weight gain in chicks fed graded level of arginine (based on Webel, Johnson, and Baker, 1998)

NC = negative control

This data on poultry complements the results for swine, again showing that endotoxin-induced energy losses quantifiably depress animal performance even in milder disease cases.

The way forward: Endotoxin mitigation

Animals suffering from endotoxemia are subject to severe metabolic dysfunctions. If they do not perish from septic shock, they are still likely to show performance losses. Moreover, they at great risk of immunosuppression caused by the immune system “overdrive.” Effective endotoxin mitigating agents can help to prevent these scenarios.

EW Nutrition’s Mastersecure Gold is not only a leading anti-mycotoxin agent; thanks to its specific components, it effectively binds bacterial toxins. An in vitro study conducted at the Hogeschool Utrecht laboratory (part of Utrecht University) evaluated the binding capacity of Mastersecure Gold on LPS compared to three different competitor products. All products were tested at two different inclusion rates. At an inclusion rate of 0.25%, only Mastersecure Gold reduced the toxin load on the solution by 37%. At 1% inclusion, Mastersecure Gold (noted as Mastersorb below) bound 75% of the toxin, while only one competitor product demonstrated any binding (10%).

Lipopolysaccharides are a constant challenge for animal production. The quantity of Gram-negative bacteria in an animal intestine is considerable; therefore, the danger of immune system over-stimulation through endotoxins cannot be taken lightly. Producers need to prioritize the maintenance of intestinal eubiosis in production animals proactively; for instance, through targeted gut health-enhancing additives based on phytomolecules and, possibly, organic acids.

Most importantly, the detrimental impact of LPS can be mitigated by using a high-performance agent such as Mastersecure Gold. To limit losses from an energy point of view yields positive results in terms of production levels and the prevention of secondary infections, preserving animal health and farms’ economic viability.

By Claudio Campanelli, EW Nutrition

References

Adib-Conquy, Minou, and Jean-Marc Cavaillon. “Compensatory Anti-Inflammatory Response Syndrome.” Thrombosis and Haemostasis 101, no. 01 (2009): 36–47. https://doi.org/10.1160/th08-07-0421.

Huntley, Nichole F., C. Martin Nyachoti, and John F. Patience. “Immune System Stimulation Increases Nursery Pig Maintenance Energy Requirements.” Iowa State University Animal Industry Report 14, no. 1 (2017). https://doi.org/10.31274/ans_air-180814-344.

Li, Jiaolong, Yongqing Hou, Dan Yi, Jun Zhang, Lei Wang, Hongyi Qiu, Binying Ding, and Joshua Gong. “Effects of Tributyrin on Intestinal Energy Status, Antioxidative Capacity and Immune Response to Lipopolysaccharide Challenge in Broilers.” Asian-Australasian Journal of Animal Sciences 28, no. 12 (2015): 1784–93. https://doi.org/10.5713/ajas.15.0286.

Mani, Venkatesh, James H Hollis, and Nicholas K Gabler. “Dietary Oil Composition Differentially Modulates Intestinal Endotoxin Transport and Postprandial Endotoxemia.” Nutrition & Metabolism 10, no. 1 (2013): 6. https://doi.org/10.1186/1743-7075-10-6.

Webel, D.M., R.W. Johnson, and D.H. Baker. “Lipopolysaccharide-Induced Reductions in Body Weight Gain and Feed Intake Do Not Reduce the Efficiency of Arginine Utilization for Whole-Body Protein Accretion in the Chick.” Poultry Science 77, no. 12 (1998): 1893–98. https://doi.org/10.1093/ps/77.12.1893.

Zachary, James F. “Chapter 4 – Mechanisms of Microbial Infections.” Essay. In Pathologic Basis of Veterinary Disease, 132–241. St Louis, MO: Mosby, 2017. https://doi.org/10.1016/B978-0-323-35775-3.00004-7.

Another tool to reduce the use of antibiotics is the use of immunoglobulins from eggs.
Trials showed that this product is effective to support a calf’s start in life and also to offer support when challenged by various forms of diarrhoea.

The main cause for calf losses during the first two weeks of life is diarrhea. In general diarrhoea is characterised by more liquid being secreted than that being resorbed. However, diarrhoea is not a disease, but actually only a symptom. Diarrhea has a protective function for the animal, because the higher liquid volume in the gut increases motility and pathogens and toxins are excreted faster. Diarrhoea can occur for several reasons. It can be caused by incorrect nutrition, but also by pathogens such as bacteria, viruses and protozoa.

Bacteria in the gut
E. coli belong to the normal gut flora of humans and animals and can be mainly found in the colon. Only a fraction of the serotypes causes diseases. The pathogenicity of E.coli is linked to virulence factors. Decisive virulence factors are for example the fimbria used for the attachment to the gut wall and the bacteria’s ability to produce toxins.

Salmonella in general plays a secondary role in calf diarrhea, however, salmonellosis in cattle is a notifiable disease. Disease due to Clostridia is amongst the most expensive one in cattle farming globally. In herbivores, clostridia are part of the normal gastro-intestinal flora, only a few types can cause serious disease. In calves, Clostridium perfringens occurs with the different types A, C, and D. Rotaviruses are the most common viral pathogens causing diarrhoea in calves and lambs. They are mainly found at the age of 5 to 14 days. Coronaviruses normally attack calves at the age of 5 to 21 days. Cryptosporidium parvum is a protozoa and presumed to be the most common pathogen causing diarrhoea (prevalence up to more than 60 %) in calves.

Undigested feed and incorrect use of antibiotics
Plant raw materials (mainly soy products) are partly used in milk replacers as protein sources. These products contain carbohydrates, that cannot be digested by calves which can lead to diarrhea. The transition from milk to milk replacer can also be a reason.

An early application of tetracyclines and neomycin to young calves can lead to a change in the villi, malabsorption and therefore to slight diarrhoea. Longer therapies using high dosages of antibiotics can also lead to a bacterial superinfection of the gut. The problem is that in a disease situation, antibiotics are often used incorrectly. The use of antibiotics only makes sense when there is a bacterial diarrhea and not due to viruses, protozoa or poor feed management. To keep the use of antibiotics as low as possible, alternatives need to be considered.

Egg powder to add immunoglobulins
In order to achieve optimal results in calf rearing two approaches are possible. Firstly, the prophylaxis approach. This is the method of choice as diarrhoea can mostly be prevented. Therefore, it is necessary to supply the calf with the best possible equipment. As antibodies are one crucial but limiting factor in the colostrum of the “modern” cow, this gap needs to be minimised.  A study conducted in Germany in 2015 demonstrated that more than 50% of the new-born calves had a deficiency of immunoglobulins in the blood. Only 41% of the calves showed an adequate concentration of antibodies in the blood (>10 mg IgG/ml blood serum). Immunoglobulins contained in hen eggs (IgY) can partly compensate for poor colostrum quality and serve as a care package for young animals. A trial was conducted with an egg powder product* on a dairy farm (800 cows) in Brandenburg, Germany. In total 39 new-born calves were observed until weaning (65^th day of life). Before birth, the calves were already divided into control and trial group according to the lactation number of their mother cow. All calves were fed the same and received four litres of colostrum with ≥ 50 mg IgG /ml on the first day of life.

Control (n=20):            no additional supplementation
Trial group (n=19):      day 1 – 5: 100 g of the egg powder product per animal per day mixed into the colostrum or milk.

It was shown that the calves in the trial group showed a significantly higher (13%) weaning weight (105.74 kg compared to 93.45 kg in the control group) and 18%  higher average daily gain (999 g compared to 848 g in the control group) (Figure 1 and Figure 2).

Support during acute diarrhea
When diarrhea occurs, the calf has to be treated. So the second approach is to find the best and quickest solution. It is not always necessary to use antibiotics, as they do not work against virus or protozoa. Egg antibodies can be an answer when combined with electrolytes as the following trial shows. On a dairy farm (550 cows) in Germany a feeding trial with a product based on egg powder and electrolytes** was conducted from December 2017 to May 2018. Two groups of calves were used. Before birth the animals were allocated into the two groups according to the calving plan and were examined from day one until weaning (77^th day of life). All calves suffering from diarrhea (38 in total, 17 in the control and 21 in the trial group) were treated as follows:

Control (n=17):            Application of electrolytes
Trial group (n=21):      50 g of the egg powder and electrolytes product twice daily, stirred into the milk replacer until diarrhea stopped.

If the diarrhea did not stop or even got worse, the animals were treated with antibiotics. It was shown that in the control group the antibiotic treatment necessary was nearly twice as long as needed in the trial group (Figure 3). This means also that nearly twice the amount of antibiotics were used. This leads to the conclusion that calves in the trial group had an improved health status compared to calves in the control group. A further result from the improved health status was an increase in performance in the trial group (Figure 4).

The average daily weight gain of the trial group was 20% higher than in the control (600 vs. 500 g per day) leading to a significantly higher weaning weight (87.8 kg) than in the control (80.7 kg).

By Dr. Inge Heinzl, Editor EW Nutrition
Published in Dairy Global (Online and Printed), 10/2018