Decoding the connection between stress, endotoxins, and poultry health

Poultry

By Marisabel Caballero, Global Technical Manager Poultry, EW Nutrition

Stress can be defined as any factor causing disruptions to homeostasis, which triggers a biological response to regain equilibrium. We can distinguish four major types of stressors in the poultry industry:

  • Technological: related with management events and conditions
  • Nutritional: involving nutritional disbalances, feed quality and feed management
  • Pathogenic: comprising health challenges.
  • Environmental: changes in environment conditions

In practical poultry production, multiple stress factors occur simultaneously. Their effects are also additive, leading to chronic stress. The animals are not regaining homeostasis and continuously deviate the use of resources through inflammation and the gut barrier-function, thus leading to microbiome alteration. As a consequence, welfare, health, and productivity are compromised.

What are endotoxins?

Bacterial lipopolysaccharides (LPS), also known as endotoxins, are the main components of the outer membrane of all Gram-negative bacteria and are essential for their survival. LPS have direct contact with the bacteria’s surroundings and function as a protection mechanism against the host’s immunological response and chemical attacks from bile salts, lysozymes, or other antimicrobial agents.

Gram-negative bacteria are part of animals’ microbiota; thus, there are always LPS in the intestine. Under optimal conditions, this does not affect the animals, because intestinal epithelial cells are not responsive to LPS when stimulated from the apical side. In stress situations, the intestinal barrier function is impaired, allowing the passage of endotoxins into the blood stream. When LPS are detected by the immune system either in the blood or in the basolateral side of the intestine, inflammation and changes in the gut epithelial structure and functionality occur.

The gut is critically affected by stress

Even when there is no direct injury to the gut, signals from the brain can modify different functions of the intestinal tract, including immunity. Stress can lead to functional disorders, as well as to inflammation and infections of the intestinal tract. Downstream signals act via the brain–gut axis, trigger the formation of reactive oxygen and nitrogen species as well as local inflammatory factors, and circulating cytokines, affecting intestinal homeostasis, microbiome, and barrier integrity.

Stress then results in cell injury, apoptosis, and compromised tight junctions. For this reason, luminal substances, including toxins and pathogens, leak into the bloodstream. Additionally, under stress, the gut microbiome shows and increment on Gram-negative bacteria (GNB). For instance, a study by Minghui Wang and collaborators (2020) found an increase of 24% in GNB and lower richness, in the cecum of pullets subjected to mild heat stress (increase in ambient temperature from 24 to 30°C).

Both these factors, barrier damage and alterations in the microbiome, facilitate the passage of endotoxins into the blood stream, which promotes systemic chronic inflammation.

What categories of stress factors trigger luminal endotoxins’ passage into the bloodstream?

Technological stress

Various management practices and events can be taken as stressors by the animals’ organism. One of the most common examples is stocking density, defined as the number of birds or the total live weight of birds in a fixed space. High levels are associated with stress and loss of performance.

A study from the Chung-Ang University in 2019 found that broilers with a stocking density of 30 birds/m2 presented two times more blood LPS than birds kept at half of this stocking density. Moreover, the body weight of the birds in the high-density group was 200g lower than the birds of the low-density group. The study concluded that high stocking density is a factor that can disrupt the intestinal barrier.

Nutritional stress

The feed supplied to production animals is designed to contribute to express their genetic potential, though some feed components are also continuous inflammatory triggers. Anti-nutritional factors, oxidized lipids, and mycotoxins induce a low-grade inflammatory response.

For instance, when mycotoxins are ingested and absorbed, they trigger stress and impair immunity in animals. Their effects start in gastrointestinal tract and extend from disrupting immunity to impairing the intestinal barrier function, prompting secondary infections. Mycotoxins can increase the risk of endotoxins in several ways:

A study conducted by EW Nutrition (Figure 1) shows an increase in intestinal lesions and blood endotoxins after a mycotoxin challenge of 200pbb of Aflatoxin B1 + 360ppb Ochratoxin in broilers at 21 days of age. The challenged birds show two times more lesions and blood endotoxins than the ones in the unchallenged control. The use of the right mitigation strategy, a product based on bentonite, yeast cell walls, and phytogenics (EW Nutrition GmbH) successfully prevented these effects as it not only mitigates mycotoxins, but also targets endotoxins in the gut.

Figure 1
Figure 1 Blood LPS and intestinal lesion score of broilers challenged with 200ppb AFB1 + 350 ppb OTA from 1 to 21 days of age without and with an anti-toxin product from EW Nutrition GmbH (adapted from Caballero et al., 2021)

Pathogenic stress

Intestinal disease induces changes in the microbiome, reducing diversity and allowing pathogens to thrive. In clinical and subclinical necrotic enteritis (NE), the intestinal populations of GNB, including Salmonella and E.coli also increases. The lesions associated with the pathogen compromise the epithelial permeability and the intestinal barrier function, resulting in translocation of bacteria and LPS (Figure 5) into the bloodstream and internal organs.

Figure 2
Figure 2 Increase in systemic LPS (vs a healthy control) after a NE challenge (adapted from Chen et al., 2015, Li et al., 2018 & Liu at al., 2018)

Environmental stress

Acute and chronic heat and cold stress increases gut permeability, by increasing intestinal oxidative stress and disrupting the expression of tight junction proteins. This results in the damage and destruction of intestinal cells, inflammation, and imbalance of the microbiota. An increased release and passage of endotoxins has been demonstrated in heat stress (Figure 3), as well as a higher expression of TLR-4 and inflammation.

Figure 3
Figure 3 Systemic LPS increase (in comparison with a non-stressed control) after different heat stress challenges in broilers: 16°C increased for 2, 5 and 10 hours (Huang et al., 2018); 9°C increased for 24 and 72 hours (Nanto-Hara et al., 2020); 10°C continuously for 3 and 10 days, and 15°C 4 hours daily for 3 and 10 days (Alhenaky et al., 2017)

 

Zhou and collaborators (2021) showed that 72 hours of low temperature treatment in young broilers increased intestinal inflammation and expression of tight junction proteins, while higher blood endotoxins indicate a disruption of the intestinal barrier. As a consequence, the stress decreased body gain and increased the feed conversion rate.

An experiment conducted by EW Nutrition GmbH with the objective of evaluating the ability of a toxin mitigation product to ameliorate heat-stress induced LPS. For the experiment, 1760 Cobb 500 pullets were divided into two groups, and each was placed in 11 pens of 80 hens, in a single house. One of the groups received feed containing 2kg/ton of the product from the first day. From week 8 to week 12, the temperature of the house was raised 10°C for 8 hours every day.

Throughout the heat stress period, blood LPS (Fig 4) was lower in the pullets receiving the product, which allowed lower inflammation, as evidenced by the lower expression of TLR4 (Fig. 5). Oxidative stress was also mitigated with the help of the combination of phytomolecules in the product, obtaining 8.5% improvement on serum total antioxidant capacity (TAC), supported by an increase in in superoxide dismutase (SOD glutathione peroxidase (GSH) and a decrease in malondialdehyde (MDH).

Fig

Figures 4-5
Figures 4 and 5 – Blood LPS and expression of toll-like receptor 4 (TLR4) in lymphocites of pullets before (wk 6) and during heat stress (wk 9 and 10). (*) indicates significant differences (P<0,05), and (‡) a tendency to be different against the control group (P<0,1).

In practice: there is no silver bullet

In commercial poultry production, a myriad stressors may occur at the same time and some factors trigger a chain of events that work to the detriment of animal health and productivity. Reducing the solution to the mitigation of LPS is a deceitfully simplistic approach. However, this should be part of a strategy to achieve better animal health and performance. In fact, EW Nutrition’s toxin mitigation product alone helped the pullets to achieve 3% improvement in body weight and 9 points lower cumulative feed conversion (Figure 6).

Figure 6
Figure 6

Keeping the animals as free of stress as possible is a true priority for poultry producers, as it promotes animal health as well as the integrity and function of the intestinal barrier. Biosecurity, good environment, nutrition and good management practices are crucial; the use of feed additives to reduce the consequences of unavoidable stress also critically supports the profitability of poultry operations.

 




Understanding the dangers of mycotoxins for breeder hens

eggs

As the producers of hatching eggs and day-old chicks, breeding hens are the backbone of the poultry industry. Hence it is common practice to pay particular attention to this valuable asset’s feed, selecting raw materials of high nutritional quality and safety. However, in any feed formulated for animals in production and reproduction, studies show that it is almost inevitable to find a certain level of mycotoxin contamination.

Mycotoxins exert toxic effects mainly on the gastrointestinal tract, liver, and kidneys and can accumulate in some tissues but also in the eggs. Mycotoxin contamination in breeder hens rations does not always lead to visible symptoms, such as when trichothecenes cause oral lesions. However, it may influence productivity, egg quality, hatchery performance, as well as chick quality and immunity. Mycotoxin risk management is thus an essential part of managing breeder hens. Mycotoxins can negatively affect eggshell quality and, as a consequence, embryonic mortality.

Mycotoxin management for breeding hens.

By Marisabel Caballero, Global Technical Manager Poultry at EW Nutrition.

 

Type of mycotoxin and exposure time determine effect on egg production

Mycotoxicosis in hens can cause reduced egg production, most likely because it causes a decrease in protein synthesis. A lower synthesis of albumin results from a degeneration of the liver tissue due to aflatoxin, ochratoxin, T2 and DON exposure. The liver then may look pale, friable and occasionally shows superficial hemorrhages.

The contamination levels at which these effects can be observed are as low as 100ppb in feed, for example, during a 21-day exposure to ochratoxin (Figure 1). With increasing levels of the toxin, production further decreases. A similar effect is observed when breeder hens are exposed to aflatoxins.

Egg production influenced by mycotoxins

Figure 1 – Effect of mycotoxins on egg production, compared to non-contaminated control (=100 %)

Egg production, however, is not the only parameter that is affected when breeding hens are exposed to mycotoxins. Earlier on in the reproductive cycle, they already impact on embryonic mortality and hatchability. These effects are potentially more severe and may even occur without any noticeable change in the number of eggs produced.

Mycotoxins’ insidious consequences for eggshell quality and embryonic mortality

The eggshell is important to protect the progeny: thin and fragile shells can increase embryonic mortality, lower embryonic weight gain and decrease hatchability. Eggshell quality is a function of the hen’s calcium and vitamin D3 metabolism. The bioavailability of calcium and of vitamin D3 depends on intestinal integrity and on the production of enzymes and transporters that aid in feed metabolism. These processes can be adversely affected by aflatoxins, DON, T2, and Fumonisins.

The gastrointestinal tract is not the only site of mycotoxin action, however. Mycotoxins such as aflatoxins and ochratoxins have nephrotoxic effects, affecting calcium metabolism and increasing its excretion via the urine, while lowering its levels in blood serum.

Moreover, mycotoxins damage the liver, which plays a central role in egg production, being responsible for vitamin D3 metabolism and the synthesis of the lipids that make up the yolk. Moreover, the synthesis of transporters for lipids, calcium, and carotenoids   ̶  important components of the egg  ̶  also takes place in the liver. When liver function is impaired, the internal and external quality of the egg declines, which, in the end, affects the production of day-old chicks.

Effects of mycotoxins on eggshell quality and embryonic mortality

Figure 2 – Effects of mycotoxins on eggshell quality and embryonic mortality

Figure 2 summarises the possible ways in which mycotoxins can negatively affect eggshell quality and, as a consequence, increase embryonic mortality. If a hen’s intestinal integrity is compromised, the utilization of nutrients decreases. Liver and kidney damage leads to a diminished availability of calcium and other nutrients necessary for egg formation. The birds’ calcium (and phosphorus) levels in the plasma are then lower and may lead to a greater mobilization of calcium from the bones. However, this response cannot be maintained and the eggs get a thinner shell.

The thickness of the eggshell influences the egg’s moisture loss and exchange with the environment during the incubation period. An eggshell of optimal quality does not allow the loss of nutrients and prevents bacterial contamination. Thinner eggshells are less able to fulfill these functions, leading to higher embryo mortality.

Effects of mycotoxins on embryonic quality

Figure 3 – Effects of mycotoxins on embryonic mortality

Figure 3 shows the effect of different mycotoxins on embryonic mortality. Incremental levels of ochratoxin or aflatoxin heighten embryonic mortality in a range from 1.5 to 7.5 times the embryonic mortality of the control group. In some cases, embryos are affected even when the hens received feed contaminated with mycotoxin levels that are within the guidelines suggested by the EFSA.

For example, an exposure to 4900ppb of DON for ten weeks increases the number of embryos with abnormalities. The causes are not entirely clear, as only traces of DON can be found in the egg. However, we do know that this mycotoxin can affect the protein synthesis at the level of the hen’s liver and therefore compromise the deposition of nutrients into the egg.

Mycotoxins’ effects on the progeny may cause long-term damage

Ochratoxin and aflatoxin can be transferred into the egg, where they exert toxicity on the embryos. This does not necessarily result in mortality. However, the chicks can suffer from a compromised immune function due to two reasons: lower transmission of antibodies from the hen and lower viability of the chickens’ immune cells, accompanied by a lower relative weight of the bursa of Fabricio and the thymus.

When both aflatoxin and ochratoxin are present in the feed, the effect on these parameters is synergistic. As a consequence of mycotoxin contamination, the animals’ immune response is impaired, which makes them more susceptible to infection. The final result could be increased early chick mortality due to a higher incidence of bacterial and viral infections.

The transmission of other mycotoxins into the egg is minimal. While this means that a direct effect on the progeny is unlikely to occur, mycotoxin contamination still has a snowball effect: we have to consider the indirect effect of a lower deposition of nutrients on chick quality.

Prevention is key: mycotoxin risk management for breeder hens

The best approach to manage mycotoxin risk is to implement an integrated strategy that includes good crop and grain storing practices, regular raw material sampling and mycotoxin evaluation and analysis. Management tools (such as MasterRisk) can help to evaluate mycotoxin interactions and to choose the best strategy for dealing with specific mycotoxin challenges.

The results of mycotoxin analyses can be used to take decisions regarding the inclusion levels of raw materials and in choosing feed additives that counteract mycotoxins. Products based on plant extracts, yeast cell walls, and clay minerals can help to stabilize a digestive system challenged by mycotoxins. They support the barrier function in the intestine, preventing the passage of mycotoxins into the bloodstream.

Phytomolecules are another piece of the puzzle: thanks to their antimicrobial, anti-inflammatory and antioxidant properties, they support liver function. This is particularly important for long-living animals prone to accumulating mycotoxins in their body tissues.

For a long time the “deleterious effects” of mycotoxins on breeder hens and “their repercussions on progeny health status and performance have not received from a scientific point of view as much attention”(Calini and Sirri, 2007) as they ought to have. However, now that the dangers of mycotoxins for breeder hens’ welfare, health and performance are better understood, it is clear that mycotoxin risk evaluation and management is central to successful poultry production.

*This article first appeared in All About Feed on 31 October 2018

 

Reference:

Photo: Hans Prinsen.

Brake, J., P. B. Hamilton, and R. S. Kittrell. “Effects of the Trichothecene Mycotoxin Diacetoxyscirpenol on Feed Consumption, Body Weight, and Oral Lesions of Broiler Breeders.” Poultry Science 79, no. 6 (June 01, 2000): 856-63 doi:10.1093/ps/79.6.856. 

Brake, J., P. Hamilton, and R. Kittrell. “Effects of the Trichothecene Mycotoxin Diacetoxyscirpenol on Egg Production of Broiler Breeders.” Poultry Science 81, no. 12 (December 01, 2002): 1807-810 doi:10.1093/ps/81.12.1807.

Bryden, Wayne L. “Mycotoxin Contamination of the Feed Supply Chain: Implications for Animal Productivity and Feed Security.” Animal Feed Science and Technology 173, no. 1-2 (2012): 134-58 doi:10.1016/j.anifeedsci.2011.12.014.

Calini, F., and F. Sirri. “Breeder Nutrition and Offspring Performance.” Revista Brasileira De Ciência Avícola 9, no. 2 (2007): 77-83 doi:10.1590/s1516-635×2007000200001. 

Hester, Patricia Y. “Improving Egg Production and Hen Health with Calcium.” In Egg Innovations and Strategies for Improvements, edited by Patricia Y. Hester, 319-29. London: Academic Press, 2017 doi:10.1016/b978-0-12-800879-9.00030-5.

Ul-Hassan, Zahoor, Muhammad Zargham Khan, Ahrar Khan, Ijaz Javed, and Muhammad Kashif Saleemi. ” Immunological status of the progeny of breeder hens kept on ochratoxin A (OTA)- and aflatoxin B1 (AFB1)-contaminated feeds.” Journal of Immunotoxicology 9, no. 4 (April 24, 2012): 381-91. doi:10.3109/1547691X.2012.675365.