by EW Nutrition USA, Inc.

In the poultry industry, Necrotic Enteritis is of great interest due to the potential detrimental growth effects it may have in a flock, even at subclinical levels⁵⁰. Coccidiostats and antibiotics have been used for a long time to get the disease-causing bacterium Clostridium perfringens under control, but with increasing antimicrobial resistance, alternative approaches are required. This article aims to give an overview of the disease and the measures against it.

Clostridium perfringens – a ubiquitous, highly resilient bacterium

Clostridium perfringens is a Gram-positive, spore-forming, anaerobic, rod-shaped bacterium⁵⁰. This encapsulated, non-motile microorganism is fastidious in growth requirements⁵⁹. Most often, complex media like cooked meat or thioglycolate broth are used as enrichment³⁰.

It was Welch and Nuttall who first identified C. perfringens in 1892 as Bacillus aerogenes capsulatus¹⁸. In Great Britain, the bacterium was commonly known as C. welchii and sometimes called Frankel’s bacillus in Germany until designated C. perfringens by Bergey¹³.

Clostridium perfringens is the causal microorganism for Necrotic Enteritis (NE)¹⁴. In humans, it is one of the most common causes of foodborne illness²⁰. The Centers for Disease Control and Prevention (CDC, 2012) estimates that nearly one million people are affected every year, making C. perfringens the third most frequent source of domestically acquired foodborne illness after Norovirus and Salmonella.

Clostridium perfringens can be found everywhere

Clostridium perfringens is found in soil, water, and other organic materials. As far as poultry facilities, C. perfringens has been isolated from litter, dust, walls, floors, fans, transportation coops, feeders, and feed⁸⁹.

Additionally, C. perfringens is found in the GI tract of broiler chickens, humans, and other mammals⁴⁷. When intestinal samples of broiler chickens were analyzed for C. perfringens, 75-95 % tested positive²⁴. Drew and co-workers¹⁰ determined that C. perfringens is usually found at ~10⁴ colony-forming units (CFU)/g of broiler digesta. These results agree with Jia et al.²⁶, who stated that C. perfringens is present at low levels in healthy poultry. In humans, investigations in different parts of the world showed a prevalence of Clostridium perfringens between 57-94%³².

Different types of Clostridium perfringens with different toxins

There are five types (A-E) of C. perfringens, which can be identified through their toxin production (see table 1). All strains produce alpha-toxin. Furthermore, Clostridium perfringens has been described to produce eight other toxins, three (delta, theta, kappa) can be lethal, but these are seldom involved in disease origin³⁷.

Table 1. Different types of Clostridium perfringens

High resilience gives an advantage against competitors

Since Clostridium perfringens is a spore-forming bacterium, it is very resilient to high temperatures, slight pH variations, and toxic chemicals^{43, 7}.

Labbe et al.³⁰ established that C. perfringens can reproduce at temperatures between 15-50 °C. Hence, proper refrigeration temperatures (below 10 °C) can be an effective means of control. The optimum range is between 37-47 °C, and at these temperatures, the mean generation time – the time required for the bacterial count to double – is approximately 10-12 minutes⁴¹. These short generation times allow the bacteria to outcompete other microorganisms that may need similar resources in a certain environment.

The optimum pH range of Clostridium perfringens is between 5.5-7.0²². However, it can grow at a pH as low as 5 and as high as 9. In live broiler chickens, the pH in the small intestine has been determined to be between 6.00-7.78.

Necrotic enteritis in poultry

The disease necrotic enteritis was first described by Parish^{45, 46} in cockerels in England. Some of the symptoms include depression, reluctance to move, ruffled feathers, somnolence, diarrhea, loss of appetite, and anorexia²¹. Mortality ranges from 0-50% ⁶ have been reported in infected flocks. Since then, virtually every area that raises poultry has reported signs of necrotic enteritis.

Clostridium perfringens – How NE unravels

As already mentioned, 10⁴ colony-forming units (CFU)/g of broiler digesta¹⁰ are normal and can be found in healthy birds. C. perfringens becomes problematic when counts reach 10⁷-10⁸ CFU/g⁶.

Necrotic enteritis is caused by types A and C of Clostridium perfringens, but normally, predisposing factors “set the stage”^{24, 48}. This could be seen in an investigation where they wanted to create a model to reproduce NE in a laboratory setting. Researchers realized that inoculation of C. perfringens alone did not cause the disease found in the field⁴⁸. Therefore, it was assessed that certain cofactors must play a significant role in the pathogenicity of C. perfringens. Williams⁵⁷ reviewed concurrent infections of coccidiosis and necrotic enteritis in chickens (Figure 1). The copious interactions of these diseases with predisposing factors, control methods, sources of infection, and disease form is a testament to the complexity of this poultry industry matter.

Coccidiosis creates access

Shane et al.⁵³ noted that several authors had considered coccidiosis to be a predisposing factor for NE. They proceeded to describe the pathogenesis of Eimeria acervulina, one of the protozoa responsible for coccidiosis in poultry. When the oocysts are ingested, they quickly attach to the intestinal wall causing lesions where the protozoa reproduce numerous times. These are the lesions to which C. perfringens attaches.

What happens in the animal?

Long et al.³³ proposed the pathogenesis for NE: First, epithelial cells are vacuolated, and the epithelium lifts off the lamina propria, which is congested and edematous. These lesions can be caused by a combination of factors like toxin production and/or, as just mentioned, coccidiosis. Clostridium perfringens cells attach to the lamina propria, where they thrive. The tissue becomes necrotic as large numbers of heterophils, a type of phagocyte, flood the foci (sites of lesions).

A combination of disease-inducing factors such as bacteria proliferation, heterophil lysis, and villus’ necrosis seem to develop quickly. The inflammation zone then becomes riddled with mononuclear cells, cells containing lymphocytes, antigen-presenting cells, and eosinophilic-staining (proteinaceous) amorphous material. This necrotizing process moves from the tip of the villi to the crypt.

Chronic version

In chronic cases, villi may be found to have multiple cysts from recurrent necrosis. In birds that overcome the disease, injured epithelial cells are replaced by newly formed reticular structures. These new cells travel from the crypt to the tip of the villi and replace the old, damaged cells. The result is a short, flat villus with a reduced surface area for nutrient absorption^{44, 45, 34}. These morphologically altered villi are the necrotic lesions found in the field and some C. perfringens challenge trials (Figure 2).

Acute form

The acute form of NE results in enlarged lesions along the gut wall, and the epithelium becomes eroded and detached; consequently, a diphtheritic membrane is formed. This yellow, green, or brownish pseudo-membrane is called the “Turkish towel,” which describes the appearance of the friable, gas-filled, foul-smelling GI tract⁵⁷.

Subclinical form

Poultry producers are not only concerned with the acute form of NE. Recent studies have shown that the disease’s subclinical form can be as detrimental as the acute illness¹⁹. Lovland and co-workers³⁵ stated that this symptomless disease is often overlooked at the farm, and the effects are only noticed at the processing facility.

Subclinical NE (SNE) can cause cholangiohepatitis, a condition where the liver is enlarged with pale reticular patterns and sometimes small, pale foci. In the United Kingdom, it was estimated that 4% of broiler carcasses and 12% of livers are condemned at processing plants due to clostridial infection; thereby, reducing profit³⁶. Moreover, sparse lesions that may be found in a case of SNE may be enough to hinder growth performance; thus, resulting in an underproductive flock³⁹.

Feeding Against Necrotic Enteritis

It has been reported that diet formulation has the greatest impact on the prevalence of C. perfringens in chicken GI tracts⁶¹. The poultry industry formulates diets on a least-cost basis, which may become problematic if nutritionists do not take into consideration the pathological consequences that some ingredients may have in the GI tracts of chickens. Every feed ingredient has a specific purpose in the diet. For instance, cereal grains are fed for their energy concentration as well as fiber. Also, some grain and animal/plant meals are used for their protein content. Since these ingredients are obtained from different sources, they are highly variable in macro and micronutrients¹.

The diet provides the conditions for proliferation

There are multiple elements that affect the proliferation of C. perfringens in chicken intestines, one of the most critical factors being diet formulation^{5, 36}. Some feed ingredients have been found to exacerbate the numbers of C. perfringens in chickens’ gastrointestinal tract. Diets formulated with wheat increased NE intestinal lesion scores compared to broiler chickens fed a corn-based diet⁴. In another study, Drew et al.¹⁰ investigated the effects of different protein sources on the intestinal populations of C. perfringens in broiler chickens. Diets were formulated to contain 230, 315, and 400 g/kg of fishmeal or soy protein concentrate (SPC). The numbers of C. perfringens in the ileum and ceca increased when the amount of protein increased from 230 to 400 g/kg.

Type of grain influences the occurrence of Clostridium perfringens

Authors have studied the effects of grain inclusion on gut microbiota, and it is well established that small cereal grains such as barley, rye, and wheat tend to increase the prevalence of C. perfringens in the GI tract. Shakouri et al.⁵² investigated the influence of barley, sorghum, wheat, and corn on counts of C. perfringens in the different intestinal segments. Corn and wheat had the lowest C. perfringens counts, followed by sorghum, while barley yielded the highest counts. These findings agree with Riddell and Kong⁵¹.

Other researchers have concluded that the increase in gut viscosity and increased chyme transit time elicit the overgrowth of C. perfringens in the intestines²⁸. Grains like wheat and barley contain high amounts of non-starch polysaccharides (NSP), which increase viscosity²⁶. Furthermore, it has been alleged that, since these grains are high in NSP, the bird cannot absorb nutrients as efficiently, thereby leaving them for microbes like C. perfringens to consume³¹.

Enzymes improve nutrient availability in the presence of C. perfringens

Shakori et al.⁵² and Jia et al.²⁶ also studied the impact of several diets with the inclusion of a blend of carbohydrases such as glucanase and xylanase. Their findings suggested that enzyme addition did not affect counts of C. perfringens in the different intestinal sections. However, they did find an improvement in growth performance. They stated that enzymes improved chyme viscosity by degrading the encapsulation of nutrients in diets.

For this reason, researchers have investigated the use of enzymes in wheat and barley-based diets on the incidence of C. perfringens in chicken intestines. Jackson et al.²⁵ studied the effect of beta-mannanase addition on flocks infected with Eimeria spp. and C. perfringens. They found that feeding this enzyme significantly reduced the impact of C. perfringens on the performance of infected flocks as well as intestinal lesion scores. Moreover, the authors explained that this might be due to beta-mannanase crossing the intestinal wall to provoke an immune response. They determined that this enzyme tended to ameliorate the symptoms of necrotic enteritis, but not significantly.

MOS may have a positive impact on immunity

Hofacre et al. ²³ found similar results when birds were fed mannan-oligosaccharides. A marked effect was only found when mannan-oligosaccharides were included along with lactic acid-producing, competitive exclusion products (probiotics).

The feed form is decisive

Feed form has also been investigated on the incidence of C. perfringens. When birds were fed whole wheat compared to ground, researchers found reduced counts of C. perfringens in the gut². These results can be extrapolated to the findings of Engberg et al.¹¹. They found that when birds were fed coarse versus fine mash or pellets, C. perfringens counts were consistently higher in flocks fed mash diets. These authors concluded that feeding pellets or whole grains increases gizzard activity, which consequently triggers hydrochloric acid production and decreases pH in the GI tract. This drop in pH of approximately 0.5 units may be responsible for decreased C. perfringens counts.

Mind the protein source

Another well-established fact is that the C. perfringens population can be affected by the type of the protein source and the inclusion rates.

Potato is worse than fish

Palliyeguru et al. ⁴² studied the inclusion of protein concentrates (potato, fish, and soy) on subclinical NE. They determined that the potato-containing diet resulted in the highest incidence of C. perfringens in the gut, followed by fish and soy. Also, the potato-containing diet had the highest activity of trypsin inhibitors and lowest lipid content. Increased trypsin inhibition does not allow for the inactivation of alpha and beta toxins produced by C. perfringens, resulting in increased intestinal wall lesions.

Fish is worse than soy due to the amino acid composition

Drew et al.¹⁰ formulated diets containing fishmeal or a soy protein concentrate at different levels. Feeding dietary fishmeal resulted in a higher incidence of C. perfringens as compared to the soy protein diet. Furthermore, with increasing levels of soy and fishmeal diets, counts of C. perfringens increased as well. A notable difference in fishmeal protein concentrate compared to the soy protein concentrate was the amino acid ratio in this experiment; the methionine and glycine ratios were 1.3 times greater in fishmeal diets. Muhammed et al.⁴⁰ determined that methionine was required for C. perfringens sporulation. This may be of interest to nutritionists since some authors have estimated that 10-20 % of synthetic amino acids are not absorbed and reach the lower intestinal tract, i.e., ceca; thereby, aiding in the proliferation of C. perfringens.

Fat source – animal fat is critical

The effects of fat sources on C. perfringens population remain largely unknown. Knarreborg et al.²⁹ studied the bacterial microflora in chicken intestines after feeding different dietary fats (soy oil and a tallow and lard mix) in rations containing antibiotic growth promoters (AGP). When soy oil was fed, C. perfringens counts were significantly lower than diets containing animal fats. The authors stated that, since plant oils contain higher amounts of unsaturated fatty acids, the chyme in birds fed oil diets would have decreased viscosity, decreasing transit time. Furthermore, an additive effect was found when soy oil was provided along with AGP, which may be due to facilitated antibiotic dispersion caused by the oil’s lipophilic properties. Knarreborg et al. (2002) investigated the effects of fat sources on C. perfringens. They found that total anaerobic counts increased with animal fat addition. However, zinc bacitracin was included in their diets, specifically targeting Gram-positive microorganisms like C. perfringens; thus, potentially biasing their results.

Antibiotics and coccidiostats in the diet – helpful, but finite

Antibiotics and coccidiostats have been commonly included in poultry diets since the mid-1940s and 1950s^{61, 58}.

Prescott et al.⁴⁹ studied the inclusion of zinc bacitracin to prevent necrotic enteritis and concluded that it successfully controlled the C. perfringens challenge. Flocks in the antibiotic treatments were able to overcome disease and perform similarly to unchallenged birds. Multiple authors have replicated these results using different antibiotics such as virginiamycin and salinomycin^{17, 3, 11}.

Improvements in flock performance with the inclusion of antibiotics and coccidiostats are well understood and omnipresent in the literature. However, the potential loss of subtherapeutic antibiotic usage in livestock in the United States due to increasing concerns over antimicrobial resistance and consumer demands makes research of viable alternatives to these compounds paramount.

So, what are your alternatives?

A lot of different approaches are possible. In general, these measures should act against Clostridium perfringens while supporting gut health.

Tested substances without the desired effects

Lastly, multiple options have been studied to control C. perfringens in poultry. Some researchers have studied the inclusion of complex carbohydrates and fibers like pine shavings, guar gum, and pectin with limited success^{4, 31}. Another popular alternative is the use of competitive exclusion-based products such as prebiotics and probiotics^{27, 16}. Still, these products failed to yield consistent results.

Other options that have been investigated are the addition of lactose and organic acids^{54, 38}. Potassium diformate did not produce lowered counts of C. perfringens. Lactose reduced C. perfringens counts but resulted in undesirable ceca characteristics including, enlargement and increased fermentation⁵⁴.

Essential oils alone or in combination may be a solution

Mitsch and coworkers³⁹ investigated the efficacy of two blends of essential oils with positive effects on the reduction of C. perfringens from the gut and feces of broilers. Gaucher and coworkers¹⁵ compared growth performance and gut health of broilers fed a conventional (anticoccidials and AGPs) vs. ABF (Coccidiosis vaccine and essential oil blends) diet. They established that livability, age at slaughter, and percentage of condemnation did not change with diet type. However, average daily weight gain and FCR were negatively affected. Furthermore, NE was more prevalent in ABF flocks.  Still, many authors agree that a multifactorial approach is necessary if antibiotics should be completely replaced by these strategies³⁶.

A contemporary study by Wati et al.⁵⁶ aimed to compare AGPs to a commercial blend of essential oils fed to broilers. Authors found that chickens fed essential oils had body weights and FCRs that were statistically similar to the AGP treatment. Moreover, both AGP and essential oil treatments had statistically lower counts of Salmonella and E. coli after an oral challenge than the control group.

Conclusion

C. perfringens is a potential pathogen found in every place poultry is raised. Therefore, we must continue to identify strategies to control the development of Necrotic Enteritis. Since antibiotics alone may not always successfully control C. perfringens and have the potential for subtherapeutic use loss in the US, a multifactorial approach must be considered and investigated. Grain size, enzymes, feed form, animal protein source, fats, and feed supplements such as essential oils can affect the proliferation of C. perfringens. Nutritionists, veterinarians, and live production personnel must come together to develop the best approach for their specific complex circumstances.

Figure 1. Interaction between coccidiosis and NE with environmental factors

Solid-line arrows are beneficial in controlling disease. Dashed-line arrows impart high disease risk factors. Double-line arrows depict major disease-risk factors. AGP, antibiotic growth promoter; CIA, chick infectious anemia; CEP, competitive exclusion product; Cp, Clostridium perfringens; IBD, infectious bursal disease; MD, Marek’s disease; NE, necrotic enteritis. (Williams, R.B. 2005)

 Figure 2. Necrotic Enteritis lesions in chicken intestines

Yellowish necrotic lesions in three intestinal samples. Intestines A and C show a few marked lesions. Intestine B shows clusters of lesions typical of the “Turkish towel” syndrome. (Source: http://www.mdpi.com/2072-6651/2/7/1913/htm. Accessed: January 14, 2021).

References

1. Bedford, M.R. 1996. Interaction Between Ingested Feed and the Digestive System in Poultry. Applied Poultry Science 5:86-95.
2. Bjerrum, L., K. Pedersen, and R. M. Engberg. 2005. The Influence of Whole Wheat Feeding on Salmonella Infection and Gut Flora Composition in Broilers. Avian Disease 49:9-15.
3. Bolder, N. M., J. A. Wagenaar, F. F. Putirulan, K. T. Veldman, and M. Sommer. 1999. The Effect of Flavophospholipol (Flavomycin^R) and Salinomycin Sodium (Sacox^R) on the Excretion of Clostridium perfringens, Salmonella enteritidis, and Campylobacter jejuni in Broilers After Experimental Infection. Poultry Science 78:1681-1689.
4. Branton, S. L., B. D. Lott, J. W. Deaton, W. R. Maslin, F. W. Austin, L. M. Pote, R. W. Keirs, M. A. Latour, and E. J. Day. 1997. The Effect of Added Complex Carbohydrates or Added Dietary Fiber on Necrotic Enteritis Lesions in Broiler Chickens. Poultry Science 76:24-28.
5. Choct, M. 2009. Managing Gut Health through Nutrition. British Poultry Science 50:9-15.
6. Cooper, K., and J. G. Songer. 2009. Necrotic Enteritis in Chickens: A Paradigm of Enteric Infection by Clostridium perfringens Type A. Veterinary anaerobes and diseases 15:55-60.
7. Craven, S. E., N. J. Stern, N. A. Cox, J. S. Bailey, and M. Berrang. 1999. Cecal Carriage of Clostridium perfringens in Broiler Chickens Given Mucosal Starter Culture^TM. Avian Diseases 43:484-490.
8. Craven, S. E., N. A. Cox, N. J. Stern, and J. M. Mauldin. 2001a. Prevalence of Clostridium perfringens in Commercial Broiler Hatcheries. Avian Diseases 45:1050-1053.
9. Craven, S. E., N. J. Stern, J. S. Bailey, and N. A. Cox. 2001b. Incidence of Clostridium perfringens in Broiler Chickens and Their Environment during Production and Processing. Avian Diseases 45:887-896.
10. Drew, M. D., N. A. Syed, B. G. Goldade, B. Laarveld, and A. G. Van Kessel. 2004. Effects of Dietary Protein Source and Level on Intestinal Populations of Clostridium perfringens in Broiler Chickens. Poultry Science 83:414-420.
11. Engberg, R. M., M. S. Hedemann, Leser, T.D., and Jensen, B.B. 2000. Effect of Bacitracin and Salinomycin on Intestinal Microflora and Performance of Broilers. Poultry Science 79: 1311-1319.
12. Engberg, R. M., M. S. Hedemann, and Jensen, B.B. 2002. The Influence of Grinding and Pelleting of Feed on the Microbial Composition and Activity in the Digestive Tract of Broiler Chickens. British Poultry Science 44:569-579.
13. Freeman, B.A. 1979. Burrows Textbook of Microbiology. W.B. Saunders Company, Philadelphia, Pennsylvania, USA.
14. Fukata, T., Y. Hadate, E. Baba, T. Uemura, and A. Arakawa. 1988. Influence of Clostridium perfringens and its Toxin in Germ-free Chickens. Research in Veterinary Science 44:68-70.
15. Gaucher, M.L., Quessy, S., Letellier, A., Arsenault, J., and M. Boulianne. 2015. Impact of a drug-free program on broiler chicken growth performances, gut health, Clostridium perfringens and Campylobacter jejuni occurrences at the farm level. Poultry Science 94: 1791-1801.
16. Geier, M. S., L. L. Mikkelsen, V. A. Torok, G. E. Allison, G. C. Olnood, M. Boulianne, R. J. Hughes, and M. Choct. 2010. Comparison of Alternatives to In-feed Antimicrobials for the Prevention of Clinical Necrotic Enteritis. Journal of Applied Microbiology 109:1329-1338.
17. George, B. A., C. L. Quarles, and D. J. Fagerberg. 1982. Virginiamycin Effects on Controlling Necrotic Enteritis Infection in Chickens. Poultry Science 61:447-450.
18. Hatheway, C. L. 1990. Toxigenic Clostridia. Clinical Microbiology Reviews 3:66-98.
19. Heier, B. T., A. Lovland, K. B. Soleim, M. Kaldhusdal, and J. Jarp. 2001. A Field Study of Naturally Occurring Specific Antibodies against Clostridium perfringens Alpha Toxin in Norwegian Broiler Flocks. Avian Diseases 45:724-732.
20. Heikinheimo, A., M. Lindstrom, and H. Korkeala. 2004. Enumeration and Isolation of cpe-Positive Clostridium perfringens Spores from Feces. Journal of Clinical Microbiology 42:3992-3997.
21. Helmboldt, C. F., and E. S. Bryant. 1971. The Pathology of Necrotic Enteritis in Domestic Fowl. Avian Diseases 15:775-780.
22. Hickey, C.S., and Johnson, M.G. 1981. Effects of pH Shifts, Bile Salts, and Glucose on Sporulation of Clostridium perfringens NTCT 8798. Applied and Environmental Microbiology 41:124-129.
23. Hofacre, C. L., T. Beacorn, S. Collett, and G. Mathis. 2003. Using Competitive Exclusion, Mannan-Oligosaccharide and Other Intestinal Products to Control Necrotic Enteritis. Journal of Applied Poultry Research 12:60-64.
24. Immerseel, F. V., J. De Buck, F. Pasmans, G. Huyghebaert, F. Haesebrouck, and R. Ducatelle. 2004. Clostridium perfringens in Poultry: an Emerging Threat for Animal and Public Health. Avian Pathology 33:537-549.
25. Jackson, M. E., D. M. Anderson, H. Y. Hsiao, G. Mathis, and D. W. Fodge. 2003. Beneficial Effect of B-Mannanase Feed Enzyme on Performance of Chicks Challenged with Eimeria and Clostridium perfringens. Avian Diseases 47:759-763.
26. Jia, W., B. A. Slominski, H. L. Bruce, G. Blank, G. Crow, and O. Jones. 2009. Effects of Diet Type and Enzyme Addition on Growth Performance and Gut Health of Broiler Chickens During Subclinical Clostridium perfringens Poultry Science 88:132-140.
27. Kaldhusdal, M., C. Schneitz, M. Hofshagen, and E. Skjerve. 2001. Reduced Incidence of Clostridium perfringens-Associated Lesions and Improved Performance in Broiler Chickens Treated with Normal Intestinal Bacteria from Adult Fowl. Avian Diseases 45:149-156.
28. Klasing, K. C. 1998. Nutritional Modulation of Resistance to Infectious Diseases. Poultry Science 77:1119-1125.
29. Knarreborg, A., M. A. Simon, R. M. Engberg, B. B. Jensen, and G. W. Tannock. 2002. Effects of Dietary Fat Source and Subtherapeutic Levels of Antibiotic on the Bacterial Community in the Ileum of Broiler Chickens at Various Ages. Applied and Environmental Microbiology 68:5918-5924.
30. Labbe, R. G. 1991. Clostridium perfringens. Journal of the Association of Official Analytical Chemists 74:711-714.
31. Langhout, D. J., J. B. Schutte, P. Van Leeuwen, J. Wiebenga, and S. Tamminga. 1999. Effect of Dietary High- and Low-methylated Citrus Pectin on the Activity of the Ileal Microflora and Morphology of the Small Intestinal Wall of Broiler Chicks. British Poultry Science 40:340-347.
32. Lindstrom, M., A. Heikinheimo, P. Lahti, and H. Korkeala. 2011. Novel Insights into the Epidemiology of Clostridium perfringens Type A Food Poisoning. Food Microbiology 28:192-198.
33. Long, J.R., Pettit, J.R., and Barnum, D.A. 1974. Necrotic Enteritis in Broiler Chickens II. Pathology and Proposed Pathogenesis. Canadian Journal of Comparative Medicine 38: 467-474.
34. Long, J. R., and R. B. Truscott. 1976. Necrotic Enteritis in Broiler Chickens III. Reproduction of the Disease. Canadian Journal of Comparative Medicine 40:53-59.
35. Lovland, A., and M. Kaldhusdal. 1999. Liver Lesions Seen at Slaughter as an Indicator of Necrotic Enteritis in Broiler Flocks. FEMS Immunology and Medical Microbiology 24:345-351.
36. McDevitt, R. M., J. D. Brooker, T. Acamovic, and N. H. C. Sparks. 2006. Necrotic Enteritis; A Continuing Challenge for the Poultry Industry. World’s Poultry Science Journal 62:221-247.
37. McDonel, J. L. 1986. Clostridium perfringens Toxins (type A, B, C, D, E). Pharmacology and Therapeutics 10:617-655.
38. Mikkelsen, L. L., J. K. Vidanarachchi, G. C. Olnood, Y. M. Bao, P. H. Selle, and M. Choct. 2009. Effect of Potassium Diformate on Growth Performance and Gut Microbiota in Broiler Chickens Challenged with Necrotic Enteritis. British Poultry Science 50:66-75.
39. Mitsch, P., K. Zitterl-Eglseer, B. Kohler, C. Gabler, R. Losa, and I. Zimpernik. 2004. The Effect of Two Different Blends of Essential Oil Components on the Proliferation of Clostridium perfringens in the Intestines of Broiler Chickens. Poultry Science 83:669-675.
40. Muhammed, S. I., S. M. Morrison, and W. L. Boyd. 1975. Nutritional Requirements for Growth and Sporulation of Clostridium perfringens. Journal of Applied Bacteriology 38:245-253.
41. Murray, P. R., K. S. Rosenthal, and M. A. Pfaller. 2009. Medical Microbiology. 6th ed. Elsevier Health Sciences, Philadelphia, PA, USA.
42. Palliyeguru, M. W. C. D., S. P. Rose, and A. M. Mackenzie. 2010. Effect of Dietary Protein Concentrates on the Incidence of Subclinical Necrotic Enteritis and Growth Performance of Broiler Chickens. Poultry Science 89:34-43.
43. Paredes-Sabja, D., Torres, J.A., Setlow, P., and Sarker, M.R. 2008. Clostridium perfringens Spore Germination: Characterization of Germinants and their Receptors. Journal of Bacteriology 190:1190-1201.
44. Parish, W. E. 1961. Necrotic Enteritis in the Fowl (Gallus Gallus Domesticus). I. Histopathology of the Disease and Isolation of a Strain of Clostridium welchii. Journal of Comparative Pathology 71:377-393.
45. Parish, W. E. 1961. Necrotic Enteritis in the Fowl. II. Examination of the Causal Clostridium welchii. Journal of Comparative Pathology 71:394-404.
46. Parish, W. E. 1961. Necrotic Enteritis in the Fowl. III. The Experimental Disease. Journal of Comparative Pathology 71:405-414.
47. Pedersen, K., L. Bjerrum, B. Nauerby, and M. Madsen. 2003. Experimental Infections with Rifampicin-resistant Clostridium perfringens Strains in Broiler Chickens Using Isolator Facilities. Avian Pathology 32:403-411.
48. Pedersen, K., L. Bjerrum, O. Heuer, D. Wong, and B. Nauerby. 2007. Reproducible Infection Model for Clostridium perfringens in Broiler Chickens. Avian Diseases 52:34-39.
49. Prescott, J. F., R. Sivendra, and D. A. Barnum. 1978. The Use of Bacitracin in the Prevention and Treatment of Experimentally-induced Necrotic Enteritis in the Chicken. Canadian Veterinary Journal 19:181-183.
50. Rehman, H., W. A. Awad, I. Lindner, M. Hess, and J. Zentek. 2006. Clostridium perfringens Alpha Toxin Affects Electrophysiological Properties of Isolated Jejunal Mucosa of Laying Hens. Poultry Science 85:1298-1302.
51. Riddell, C., and X. Kong. 1992. The Influence of Diet on Necrotic Enteritis. Avian Diseases 36:499-503.
52. Shakouri, M. D., P. A. Iji, L. L. Mikkelsen, and A. J. Cowieson. 2008. Intestinal Function and Gut Microflora of Broiler Chickens as Influenced by Cereal Grains and Microbial Enzyme Supplementation. Journal of Animal Physiology and Animal Nutrition 93:647-658.
53. Shane, S. M., J. E. Gyimah, K. S. Harrington, and T. G. Snider. 1985. Etiology and Pathogenesis of Necrotic Enteritis. Veterinary Research Communications 9:269-287.
54. Takeda, T., T. Fukata, T. Miyamoto, K. Sasai, E. Baba, and A. Arakawa. 1995. The Effects of Dietary Lactose and Rye on Cecal Colonization of Clostridium perfringens in Chicks. Avian Diseases 39:375-381.
55. Tschirdewahn, B., S. Notermans, K. Wernars, and F. Untermann. 1991. The Presence of Enterotoxigenic Clostridium perfringens Strains in Faeces of Various Animals. International Journal of Food Microbiology 14:175-178.
56. Wati, T., Ghosh, T., Syed, B., and S. Haldar. 2015. Comparative efficacy of a phytogenic feed additive and an antibiotic growth promoter on production performance, caecal microbial population and humoral immune response of broiler chickens inoculated with enteric pathogens. Animal Nutrition 1(2015): 213-219.
57. Williams, R.B. 2005. Intercurrent Coccidiosis and Necrotic Enteritis of Chickens: Rational, Integrated Disease Management by Maintenance of Gut Integrity. Avian pathology 34(3):159-180.
58. Williams, R. B., R. N. Marshall, R. M. La Regione, and J. Catchpole. 2003. A New Method for the Experimental Production of Necrotic Enteritis and its Use for Studies on the Relationships Between Necrotic Enteritis, Coccidiosis and Anticoccidial Vaccination of Chickens. Parasitology Research 90:19-26.
59. Wise, M. G., and G. R. Siragusa. 2005. Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR. Applied and Environmental Microbiology 71:3911-3916.
60. Wiseman, R.W., Bushnell, O.A., and Rosengerg, M.M. 1956. Effects of Rations on the pH and Microflora in Selected Regions of the Intestinal Tract of Chickens. Poultry Science 35:126-132.
61. Yegani, M., and D. R. Korver. 2008. Factors Affecting Intestinal Health in Poultry. Poultry Science 87:2052-2063.

Older laying birds are still a valuable asset, as long as they are managed for performance and productivity. Eggshell quality is one of the elements that, without proper management, can quickly deteriorate. It is therefore essential that the egg producer takes into account all the necessary elements for the formation of high-quality eggs.

The eggshell, in a nutshell

The eggshell represents ten percent of the entire egg, by weight[i]. For instance, a 60-gram egg contains approximately 6 g of shell. Out of this particular shell, approximately 95% is CaCO₃[ii], with a total of 2.3 g of Calcium (Ca).

But where does the calcium in the eggshell come from?

The Ca required for the eggshell is obtained, in variable proportions, directly from the feed or water additives (absorbed from the gut and transported via the blood to the shell gland), or from the bone (resorbed by osteoclasts and the Ca transported to the blood to the shell gland).

Maintaining eggshell quality and bone calcium: Mission Impossible?

Eggshell quality is often negatively correlated to bone strength[iii], most probably because body calcium is redirected to the shell to the detriment of the bones and the other way around. This impacts the long-term health of the skeleton; however, modern laying hens can maintain shell quality while preserving bone mineralization[iv].

60 to 75% of shell Ca is derived from the diet on shell-forming days

Approximately 60 to 75% of shell Ca is derived directly from the diet on shell-forming days[v]. This means that the greater the proportion of Ca coming directly from the feed or water additives, the better the eggshell quality can be. Therefore, the factors that can improve shell quality will also reduce the need to mobilize bone Ca and can also help to maintain skeletal health.

In old laying birds, generally after peak production, the ability to deposit Ca onto the shell remains relatively constant[vi], so an increase in egg size after peak production will tend to result in reduced shell quality. Dietary requirements for Ca tend to increase and those for phosphorus (P) tend to decrease as hens age.

Also, as hens age, the efficiency of Ca metabolism decreases[vii]. Increases in dietary Ca and a widening of the Ca:available P ratio are intended to counter this issue. Excess dietary P can also reduce shell quality[viii].

Because of its importance in Ca and P absorption from the gut, adequate dietary vitamin D activity must also be provided[ix]. Feeding of the vitamin D metabolite 25-OH vitamin D3 can help to maintain skeletal and shell quality in high-producing laying hens[x].

Ca metabolism is a complex game

Ca metabolism is regulated by various hormones such as calcitonin, 1,25-dihydroxyvitamin D3 (calcitriol), and parathyroid hormone. Estrogen, androgens, and prostaglandins also appear to have an important role in avian Ca metabolism.

Source: Ricardo (2008)

Egg formation and Ca requirements

Source

A hen ovulates approximately 15 to 75 minutes following oviposition[xi], and the ovum takes approximately 4.25 hours to reach the shell gland[xii], at which point calcification takes approximately 17 hours[xiii]. Hens generally lay eggs in the morning and early in the afternoon[xiv]. The hen can use the Ca and P made available through diet to recover medullary bone losses during the next 5 hours after oviposition.

Once the ovum reaches the shell gland, the demand for calcium naturally increases greatly as eggshell formation progresses. The highest eggshell mineral accretion takes place 5 – 15 hours after the egg enters the shell gland[xv], which normally happens later in the afternoon and during the night preceding egg laying.

Hourly Ca requirements for eggshell calcification

Ca dietary requirements vary with species, age, breeding status, and dietary levels of vitamin D. Egg-laying birds and growing birds require more Ca than adult non-breeding birds.

Common eggshell quality problems and causes

In many cases, the source of eggshell problems can be detected by recognizing the specific markers. For instance, cracked, soft-shelled or corrugated eggs can be caused by saline water, or the impact of mycotoxins; shell-les eggs can be caused by improper amounts of Ca, P, Mn or vitamin D3, as well as by infectious bronchitis or Newcastle disease IB. However, among the main causes of eggshell quality issues is heat stress.

In hot temperatures, increased respiration rates can cause an increase in CO₂ loss. The reduction of the pool of bicarbonate ions can result in respiratory alkalosis and an increase in blood pH[xvi]. A reduction in bicarbonate ions in the shell gland reduces the formation of CaCO₃ and decreases shell quality.

Under heat stress, birds will also tend to decrease their feed intake during the day to reduce diet-induced thermogenesis. Calcium intake is therefore also reduced, and shell quality decreases as a consequence.

3 solutions for eggshell quality in older layers

Midnight feeding in hot climates

At midnight, when temperatures are typically cooler, the addition of one to two hours of light can help the birds increase feed consumption[xvii]. Midnight feeding can also have the benefit of providing a dietary source of Ca to support eggshell formation during the night and reduce reliance on bone reserves[xviii].

Nutrition supplements

Along with Calcium, some micro-minerals can also influence eggshell quality. Zinc, Manganese and Copper act as cofactors of enzymes involved in the mineralization process during eggshell formation. Although European Union legislation restricts the use of high levels of these minerals, several studies in layers indicate increased egg shell resistance by increasing the dietary concentrations of microminerals. Using organic forms of Zinc, Manganese and Copper appears to be an alternative way to increase the absorption of these minerals, as organic forms appear to be more digestible than inorganic forms. Considering the high cost of organic minerals, a mix of organic and inorganic forms of critical minerals could be a better option.

Liquid Ca supplements

If a hen is fed a diet containing only a small-particle Ca sources, such as finely ground limestone, the intestine will be deprived of a source of Ca during the night, when demand for Ca is highest. At that point, the hen will be entirely reliant on bone Ca to support eggshell formation. A combination of Ca supplementation through water additives can be a good alternative as readily available Ca to the hen to support high Ca requirements during the late afternoon and through the night. Liquid Ca additives also offer further precise and user-friendly    application.

Stimuvital IP: a liquid solution from EW Nutrition

Stimuvital IP (formerly Shellimprover) is a liquid nutritional additive for laying hens, supporting the quality of eggshells and bone health. It contains a cocktail of Ca and vitamins whose benefits in laying birds are well proven through field studies, existing literature, and years of market experience.

Benefits proven in Australia field trial

22,500 layer birds were split into two equal-size groups, one of which (11250 birds) was supplemented with Stimuvital IP for 3 days every two weeks, starting from age 53^rd to 63^rd week. Improvements in eggshell thickness and strength could be noticed after the application of Shellimprover. Egg weight was consistent in Stimuvital IP -supplemented birds.3 days every fortnight by using the Easy@ system. In total, the 11250 birds received (2 (feed lines) x 3 (times per days) x 265ml x 3 (days) x 6 (week 53, 55, 57, 59, 61, 63) 28620mll of Stimuvital IP.

Benefits proven in China field trial

The field trial was carried out on a commercial layer farm. A control group and Stimuvital IP (Shellimprover) group had 50,000 birds each. Stimuvital IP was supplemented for 3 days every two weeks, starting from age 57^th to 62^nd week. The Stimuvital IP supplementation improved eggshell quality, including eggshell thickness, laying rate, and number of saleable eggs during the trial period.

Optimizing quantity, quality, and overall profitability for layer producers

Ca concentration in the blood is controlled by many interacting feedback loops that involve Ca, phosphate, PTH, vitamin D₃, and calcitonin. Supplementation of Vitamin D₃ can help maintain skeletal and shell quality in high-producing laying hens[xxiv].

Stimuvital IP offers an essential cocktail that caters to the additional requirements of Ca and vitamins in older laying birds. It thus supports Ca metabolism and eggshell quality. And, in the end, better eggshell quality reduces broken egg percentage and optimizes the number of salable eggs and profitability for layer producers.

Notes

[i] Pelicia et al., 2009; Bello and Korver, 2019

[ii] Nys et al., 2004

[iii] Orban and Roland Sr, 1990

[iv] Bello and Korver, 2019

[v] Driggers and Comar, 1949

[vi] Roland Sr et al., 1975

[vii] Wistedt et al., 2019

[viii] Miles et al., 1983

[ix] Wen et al., 2019

[x] Silva, 2017; Akbari Moghaddam Kakhki et al., 2019

[xi] Beuving and Vonder, 1981

[xii] Roberts, 2004

[xiii] Hincke et al., 2012

[xiv] Samiullah et al., 2016; Hunniford et al., 2017

[xv] Hincke et al., 2012

[xvi] Franco-Jimenez et al., 2007

[xvii] van Staaveren et al., 2018

[xviii] Harms et al., 1996

[xix] Chowdhury, 1990

[xx] Leach and Gross, 1983

[xxi] Zhang et al., 2017

[xxii] Atteh and Leeson, 1983

[xxiii] Atteh and Leeson, 1985

[xxiv] Silva, 2017; Akbari Moghaddam Kakhki et al., 2019

Full references are available upon request.

Dysbacteriosis has been defined as the presence of a qualitatively and/or quantitatively abnormal microbiota in the proximal parts of the small intestine. This abnormal microbiota produces a cascade of reactions in the gastrointestinal tract, including reduced nutrient digestibility and impaired intestinal barrier function, increasing the risk of bacterial translocation and inflammatory responses (Panneman, 2000; Van der Klis, 2000 and Lensing, 2007).  Dysbacteriosis is not a specific disease but a secondary syndrome. Along the entire GI tract, there is a diverse microbial community comprised of bacteria, yeasts, archaea, ciliate protozoa, anaerobic fungi, and bacteriophages, commonly referred to as the intestinal microbiota.

Dysbacteriosis is an imbalance in the gut microbiota as a consequence of an intestinal disruption. The impact of dysbacteriosis can be separated into economic and welfare issues (Bailey, 2010). Dysbacteriosis can lead to very wet litter and caking issues. The prolonged contact of broilers with the caked litter can result in painful ulceration of the feet and hocks (pododermatitis and hock-burn), leading to a serious welfare issue and degradation of the carcass.

Apart from these issues, a major economic impact comes from reduced growth rates, FCR, and increased veterinary treatment costs (Kizerwetter-Świda and Binek, 2008).

Causes of dysbacteriosis

It is believed that both non-infectious and infectious factors can play a role in dysbacteriosis (DeGussem, 2007).

Non-infectious causes are:

• Diet
• Brooding
• Biosecurity
• Risk periods
• Environmental conditions

Diet

Intestinal bacteria derive most of their energy from dietary compounds. Thus, diet has a major influence over the bacterial populations (Apajalahti et al., 2004). Any change in feed and feed raw materials, as well as the physical quality of feed, influence the balance of the gut microbiota. Processing significantly affects the characteristics of the feed as a substrate for the bacterial community. Perhaps the temperature and pressure of the conditioning process give its characteristic signature to the bacterial community structure.

Inappropriate brooding conditions

The provision of optimal brooding conditions is essential for ensuring optimal gut microbiota development. Birds receiving appropriate brooding develop a gut that performs well and has a greater capacity to cope with the challenges of the broiler shed. Early access to feed and water is crucial. One of the most critical factors for the occurrence of dysbacteriosis is the lack of digesta. The microbiota can change in a period of hours when nutrients are not present. The quality of water is also essential to maintain normal intestinal function and digesta pH.

Faulty biosecurity

If clean-out and disinfection procedures are improperly conducted, pathogens will be introduced into the poultry shed, and exposure to these pathogens will influence gut health and development. It has been proven that litter management regimes affect chicken gastrointestinal tract (GIT) and microbiota (Wang et al., 2016)

Risk periods

There are times during poultry production when the bird will be challenged, for example, during feed changeovers, vaccination handling and transportation, overcrowding, or placement in new housing. During these periods, the gut microbiota can fluctuate and, in some cases, if management is sub-optimal, dysbacteriosis can occur.

Environmental conditions

Achieving optimal environmental conditions will promote good gut health. Any perturbation in gastroenteric physiology or immunity of the bird, caused by temperature stress or other environmental discomforts, can cause dysbacteriosis and/or enteritis. These are associated with lower absorption of nutrients by the host. Suzuki et al. 1983 demonstrated that overcrowding and heat stress, very commonly seen in intensive poultry farming, has a significant impact on the microbiota of chickens.

Infectious agents that potentially play a role in dysbacteriosis

• Mycotoxins
• Eimeria spp.
• Clostridium perfringens
• Other bacteria producing toxic metabolites

Mycotoxins

Many mycotoxins can stimulate the secretion of several antimicrobial molecules, which have positive effects on the maintenance of intestinal homeostasis. Fumonisins inhibit the growth of fungi, Fusarium toxins exhibit different antimicrobial defensive mechanisms, and aflatoxins exhibit a moderate antimicrobial activity against Escherichia coli, Bacillus subtilis, and Enterobacter aerogenes [Bevins et al. 1999 and Wan et al.2013]. Mycotoxins such as aflatoxins, trichothecenes, zearalenone, fumonisin, and ochratoxin can alter the normal intestinal functions, such as the barrier function and nutrient absorption. Some mycotoxins, like trichothecenes and ochratoxin, affect the histomorphology of the intestine (Winnie et al., 2018). Mycotoxicosis changes the population equilibrium, which can lead to dysbacteriosis.

Eimeria spp.

Coccidiosis caused by Eimeria spp. in chickens appears to be one of the principal destabilizing agents, causing the destruction of enterocytes and affecting the integrity of the intestinal mucosa and wall. The lesions that it causes, the inflammatory process, the reduced absorption and consequent excess of nutrients in the lumen all contribute to the proliferation of certain groups of bacteria. This situation clearly predisposes birds to intestinal dysbacteriosis and/or bacterial enteritis, and in particular to necrotic enteritis.

Clostridium perfringens

Clostridium perfringens is a natural part of the habitat in the hindgut that is not dangerous under normal circumstances. If it multiplies, the bacterium produces toxic substances that damage the intestinal mucosa and cause a condition called necrotic enteritis.  The disease is characterized by necrosis and inflammation of the GIT. Without treatment, this can escalate to perforation of the intestines, hemorrhages, and eventual death from septic shock.

Signs and consequences of dysbacteriosis

Dysbacteriosis can have profound effects on the host. Dysbacteriosis alters the GIT environment and favors the growth of pathogenic bacteria. Pathogenic bacteria produce toxins that increase intestinal motility or cause alterations in the amounts of mucus produced or in its composition. They also result in modifications of gastric acidity, reduction in the production of bacteriostatic peptides in the pancreas, and reduced immunoglobulin (IgA) secretion.

Toxins released by entero-pathogens damage intestinal villi, resulting in focal ulcerations of the mucosa, tissue necrosis, and shifts in gut microorganism numbers and metabolism. The costliest condition for animal production is the chronic inflammatory response of the animal to constant minor dysbacteriosis. These chronic responses can reduce weight gain and cause low feed conversion efficiency. Coccidiosis infections and any other enteric disease can be aggravated when dysbacteriosis is prevalent. Generally, animals with dysbacteriosis have high concentrations of Clostridium that generate more toxins, leading to necrotic enteritis.

In broilers, the syndrome is generally seen between 20 and 30 days of age (Wilson et al., 2005). Clinically, the main signs are:

• pale, glistening or orange droppings with undigested food particles
• wet and greasy droppings and hence dirty feathers
• sometimes foamy caecal droppings
• reduced physical activity
• increased water intake
• decrease in feed intake with a check in weight or reduced gain rates
• increased feed conversion

(Wilson et al., 2005; De Gussem, 2007)

Wet litter is also a general outcome of dysbacteriosis that may affect the air quality of the house, leading to a higher incidence of respiratory problems.

Additionally, foodborne pathogens such as Salmonella spp. and E.coli proliferate more in the dysbiotic intestine and can become persistent residents of the hindgut.

At necropsy, the main observations are

• a thin, fragile, often translucent intestinal wall
• watery or foamy intestinal contents
• frequent orange mucus and undigested particles in the intestines
• ballooning of the gut
• intestinal inflammation

(Pattison, 2002; De Gussem,2007)

Prevention of dysbacteriosis

The most important factors to prevent dysbacteriosis are

• Minimizing environmental stress
• Maintaining good water quality
• Improving feed digestibility
• Avoiding antinutritional factors, mycotoxins, and rancidity
• Feed additives that could modulate microbial component and avoid dysbacteriosis

Growth-promoting antibiotics are well known for the inhibition of undesired microbiota and the negative effects of their metabolites, and selection for beneficial bacteria. However, the adverse result is that they diminish the natural diversity of the gut microbiota. Antibiotics can also result in animals developing bacterial resistance.

Other products have been proposed as alternatives to growth promotion, taking into consideration the increasing bacterial resistance to some antibiotic categories.

Alternate feed additive technologies that have a promising role in controlling dysbacteriosis are:

• Probiotics
• Prebiotics
• Enzymes
• Organic acids
• Essential oils and phytomolecules

Probiotics

The post-hatch period is very critical for the chicks’ intestine development. Exposure to the environment in hatchery and farm affects microbial colonization in the intestine tract. The use of selective probiotics in day-old chicks at the hatchery and on the farm immediately after placement in broiler house reduces the risk of dysbacteriosis. Probiotics work by competitive exclusion, thereby prevent the colonization of potentially pathogenic bacteria. Probiotics prevent enteric diseases, improves intestine development and digestion process.

The benefits include enhanced growth and laying performance, improved gut histomorphology, immunity, and an increase in beneficial microbiota (Rajesh Jha et al., 2020)

Prebiotics: Mannan Oligosaccharide

(MOS) mimics the properties of the cells on the gut wall to attract and bind with harmful bacteria. Rather than allowing the bad bacteria to attach to the gut wall, the MOS acts as a sticky sponge, clearing up the harmful bacteria and removing them from the digestive system. MOS play an important role in gut functionality and health, through enhanced nutrient digestibility and improved gut barrier function and local defenses. MOS is also related to long villi and shallow crypts in the intestine, so a larger surface area helped with the absorption of nutrients and improved animal performance (Chand et al., 2016b)

Enzymes

Careful choice of feed enzymes will reduce nutrients available for pathogenic bacterial growth and improve gut health. Bacterial Xylanase is showing promise by digesting both soluble and insoluble arabinoxylans and reducing the viscosity of intestinal content. It maintains gut motility, improves nutrients digestibility, and impairs the growth of pathogenic bacteria in the hindgut.

Organic acids

Organic acids ameliorate the conditions of the GIT through the reduction of GIT pH, promoting proteolytic enzyme activity, intensifying pancreatic secretions. They encourage digestive enzyme activity and nutrient digestibility. Organic acids are creating stability of the microbial population by stimulating the growth of beneficial bacteriaPapatisiros et al., 2013).

Phytomolecules

Multiple scientific studies have proven the positive effects of phytomolecules (also known as phytogenics or secondary plant compounds) on the gut health of livestock animals. These substances support digestion and improve the utilization of nutrients. This results in higher daily weight gain and better feed conversion. In addition, phytomolecules have a proven antimicrobial effect, based on different biological modes of action.

EW Nutrition offers standardized phytomolecule-based solutions (Activo and Activo Liquid) that positively influence gut health and subsequent performance parameters in poultry. In scientific studies, the Activo product line has shown a positive effect on gut pathogenic bacteria, reducing necrotic enteritis (Fig 1) and improving production performance.

Conclusion

Dysbacteriosis can have profound effects on the host. Acute dysbacteriosis can result in the proliferation of pathogenic microorganisms that become enteropathogenic. Pathogenic bacteria can produce toxins and metabolites that increase gut motility, increase fermentation with gas production, change gut pH, irritate the mucosa, cause inflammation, and increase mucous secretion. This process reduces the digestibility and absorption of nutrients.

Maintaining the equilibrium of the gut ecosystem is key to avoiding dysbacteriosis. Improving feed digestibility and using feed additives that modulate gut microflora help to maintain more stable gut ecosystems, even during periods of intestinal stress preventing dysbacteriosis. Effective prevention and control of dysbacteriosis help increase poultry operations’ economic profitability by way of improved performance, health, and welfare, and reduce foodborne pathogens and environmental impact of poultry production.

References

Apajalahti, J., Kettunen, A., and H. Graham. 2004. Characteristics of the gastrointestinal microbial communities, with special reference to the chicken. World Poultry Sci J 60:223- 232.

Bailey, Richard A. 2010. Intestinal microbiota and the pathogenesis of dysbacteriosis in broiler chickens. PhD thesis submitted to the University of East Anglia. Institute of Food Research, United Kingdom.

Bevins, C. L.; Martin-Porter, E.; Ganz, T. Defensins and innate host defence of the gastrointestinal tract. Gut, 1999, 45, 911–915.

De Gussem , M. 2007. Coccidiosis in poultry: review on diagnosis, control, prevention and interaction with overall gut health . In Proceedings of the XVI European  Symposium on Poultry Nutrition (pp. 160 169 . Strasbourg , France.

Gurrre, Philippe. 2020. Review Mycotoxin and Gut Microbiota Interactions. Toxins, 12, 769.

Jha, Rajesh, Razib Das, Sophia Oak, and Pravin Mishra, 2020. Probiotics (Direct-Fed Microbials) in Poultry Nutrition and Their Effects on Nutrient Utilization, Growth and Laying Performance, and Gut Health: A Systematic Review. Animals (Basel). 10(10): 1863.

Kizerwetter-Świda, M., and M. Binek. 2008. Bacterial microflora of the chicken embryos and newly hatched chicken. Journal of Animal and Feed Sciences 17:224-232

Panneman, H. 2000 . Clostridial enteritis/dysbacteriosis, fast diagnosis by T-RFLP, a novel diagnostic tool. In Proceedings of the Elanco Global Enteritis Symposium. Cork Ireland.

Papatisiros VG, Katsoulos PD, Koutoulis KC, Karatzia M, Dedousi A, Christodoulopoulos G. Alternatives to antibiotics for farm animals. CAB Rev Ag Vet Sci Nutr Res. (2013) 8:1–15. doi: 10.1079/PAVSNNR20138032.

Pui-Pui, Winnie, and Sabran Mohd-Redzwan. 2018. Mycotoxin: Its Impact on Gut Health and Microbiota. Frontiers in Cellular and Infection Microbiology, 8:60.

Rebel, J.M.J., Balk, F.R.M., Post, J., Van Hemert, S., Zekarias, B. and Stockhofe, N. 2006. Malabsorption syndrome in broilers. World’s Poultry Science Journal, 62: 17–29.

Saeed, Mohammad, Fawwad Ahmad, Mohammad Asif Arain, Mohamed E Abd El-Hack, Mohamed Emam, Zohaib Ahmed Bhutto and Arman Moshaven, 2017. Use of Mannen – Oligosaccharides (MOS) As a Feed Additive in Poultry Nutrition. J. World Poult. Res. 7(3): 94-103.

Suzuki, K., R. Harasawa, Y. Yoshitake, and T. Mitsuoka. 1983. Effects of crowding and heat stress on intestinal flora, body weight gain, and feed efficiency of growing rats and chicks. Nippon Juigaku Zasshi 45:331-8.

Van der Klis, J.D. and Lensing, M. 2007. Wet litter problems relate to host–microbiota interactions. World Poultry, 23: 20–22.

Wan, M. L.; Woo, C. S.; Allen, K. J.; Turner, P. C.; El-Nezami, H. Modulation of porcine-defensins 1 and 2 upon individual and combined fusarium toxin exposure in a swine jejunal epithelial cell line. App. l. Environ. Microbiol., 2013, 79(7), 2225-2232

Wang L, Lilburn M, Zhongtang Y. 2016. Intestinal microbiota of broiler chickens as affected by litter management regimens Front. Microbiol (2016).

Wilson, J., Tice, G., Brash, M.L. and St Hilaire, S. 2005. Manifestations of Clostridium perfringens and related bacterial enteritides in broiler chickens. Worlds Poultry Science Journal, 61: 435–449.

Lutein belongs to the group of carotenoids, which is divided into carotenes and xanthophylls. Lutein, chemically expressed as “3,3’-dihydroxy-α-carotene”, is a xanthophyll always accompanied by its isomer zeaxanthin. It is synthesized out of two α-carotenes through hydroxylation.

Lutein provides benefits for animals and humans

Lutein has antioxidant protective properties

Under normal conditions, the cells in the animal and human organism control ROS (reactive oxygen species) levels. Usually, there is a balance between the generation of ROS and their elimination by scavenging systems. However, the high performance levels in modern animal production can easily lead to high ROS levels, translated into oxidative stress and leading to cell damage. Cell damage contributes to the generation of cancer and early aging in humans. In animals, the negative impact of oxidative stress can be responsible for lower performance and inferiority of meat and eggs.

Antioxidants stop ROS by taking up their energy

Through the uptake of energy, molecules can get into an excited state. One example is singlet excited oxygen, a highly reactive form of oxygen able to destroy proteins, lipids, and DNA. Carotenoids can intervene in this process: by exchanging electrons, the singlet excited oxygen gets neutralized, and the carotenoid gets into this excited state with higher energy. Once able to release this energy as heat into the environment, the carotenoid gets back to its normal state and can once again start acting as an antioxidant.

In this way, carotenoids, including lutein, ‘quench’ the energy of excited molecules and prevent the adverse effects of ROS (reactive oxidative substances).

Antioxidant properties profitably used

The antioxidant character of lutein plays an important role in the treatment or prophylaxis of macular degeneration in humans (Landrum & Bone, 2001). There is also evidence that lutein can be used to improve the visual and retinal function in dogs (Wang et al., 2016). In the eye, lutein and zeaxanthin, occurring in the retina and the macula, neutralize free radicals produced due to the ultraviolet light and thereby prevent damage to the macula.

Further possible applications are against cardiovascular diseases (Dwyer et al., 2001)  and various types of cancer (e.g., breast cancer, Gong et al., 2018).

Lutein is important in infant nutrition

Lutein and its isomer zeaxanthin are the two primary carotenoids found in human milk (Giordano and Quadro, 2018). Stringham and co-workers (2019) postulate that lutein plays an important role in children’s visual and cognitive development/optimization. They report that a lutein supplementation of the mother can lead to a higher concentration of this substance in the milk and, consequently, in the child’s plasma (Sherry et al., 2014). In dairy cows, an increased level of lutein in the milk can also be observed (Xu et al., 2014), suggesting that lutein could also be essential in calf development.

Lutein stimulates the immune system

Another benefit of lutein is its positive influence on the immune system.

On the one hand, lutein stimulates the production of antibodies. In dogs, Guimarães Alarça et al. (2016) could show an increase of CD4+ and CD8+ T-lymphocyte subtypes. Kim et al. (2000) demonstrated the increase of lymphocytes and cells expressing CD5, CD4, CD8, and major histocompatibility complex class II (MHC II) molecules. Bédécarrats and Leeson (2006) provoked a higher secondary antibody response to infectious bronchitis vaccination in laying hens.

Besides, lutein acts as an anti-inflammatory agent, as shown in vitro by Chao et al. (2015) and in broiler chickens by Moraes and team (2016).

Lutein improves the attractivity of poultry products

In the marketing of poultry products, appearance and color are of central importance for evaluating quality. Egg yolk coloration is to a large extent a matter of regional preferences, however it is clear that an egg with a yolk that does not have the typical color is classified as inferior by the consumer. In areas with traditional corn growing, a white-skinned chicken is not commercially viable. Even when pullets are bought, the shanks and beaks should be yellow.

The use of xanthophylls like lutein and zeaxanthin enables producers to safely control the color of the egg yolk and of the broiler skin. It also leads to a healthy color of the shanks and beaks of the birds.

Lutein in a nutshell

Lutein is a true all-rounder: a substance that delivers benefits across the board. In plants, it helps fruits and petals become attractive for insects and other animals. It positively influences the animal, acting as an antioxidant, promoting infant development, and stimulating the immune system. As a pigment, it makes poultry and poultry products look more attractive to the consumer. Through its presence in eggs and milk, lutein provides clear and clean benefits to both animals and humans.

References

Bédécarrats, G.Y. and S. Leeson. “Dietary lutein influences immune response in laying hens.”  J. Appl. Poult. Res. 15 (2006): 183–189.

https://doi.org/10.1093/japr/15.2.183

Chao, Shih-Chun, Tommaso Vagaggini, Chan-Wei Nien, Sheng-Chieh Huang, and Hung-Yu Lin. “Effects of Lutein and Zeaxanthin on LPS-Induced Secretion of IL-8 by Uveal Melanocytes and Relevant Signal Pathways.” Journal of Ophtalmology, vol. 2015 Article ID 152854 (2015): 7 pages. https://doi.org/10.1155/2015/152854

Dwyer, James H., Mohamad Navab, Kathleen M. Dwyer, Kholood Hassan, Ping Sun, Anne Shircore, Susan Hama-Levy, Greg Hough, Xuping Wang, Thomas Drake, C. Noel Bairey Merz, and Alan M. Fogelman. “Oxygenated Carotenoid Lutein and Progression of Early Atherosclerosis.” Circulation (American Heart Association) 103, no. 24 (2001): 2922-2927.

https://doi.org/10.1161/01.CIR.103.24.2922

Gong, Xiaoming, Joshua R. Smith, Haley M. Swanson, and Lewis P. Rubin. “Carotenoid Lutein Selectively Inhibits Breast Cancer Cell Growth and Potentiates the Effect of Chemotherapeutic Agents through ROS-Mediated Mechanisms.” Molecules 23 no. 4(2018): 905.

http://dx.doi.org/10.3390/molecules23040905

Guimarães Alarça, Laís, Fabiane Yukiko Murakami, Ananda Portella Félix, Everton Luis Krabbe, Simone Gisele de Oliveira, Sebastião Aparecido Borges da Silva. “Dietary lutein supplementation on diet digestibility and blood parameters of dogs.” Cienc. Rural 46 no.12 (2016)

http://dx.doi.org/10.1590/0103-8478cr20151493

Kim, Hong Wook, Boon Chew, Teri Ann S Wong, Jean Soon Park, Bor-Chun Weng, Katherine M Byrne, Michael G Hayek, and Gregory A. Reinhart. “Dietary lutein stimulates immune response in the canine.” Veterinary Immunology and Immunopathology 74 no. 3-4 (2000): 315-327.

https://doi.org/10.1016/S0165-2427(00)00180-X

Landrum, J. T. and R.A. Bone. “Lutein, zeaxanthin, and the macular pigment.” Archives of Biochemistry and Biophysics 385 no. 1 (2001): 28–40.

https://doi.org/10.1006/abbi.2000.2171.

Moraes, M. L., A. M. L. Ribeiro, E. Santin, and K. C. Klasing. “Immunology, health, and disease: effects of conjugated linoleic acid and lutein on the growth performance and immune response of broiler chickens.” Poultry Science 95 (2016): 237–246.

http://dx.doi.org/10.3382/ps/pev325

Ochoa Becerra, Mario, Luis Mojica Contrerasa, Ming Hsieh Loa, Juan Mateos Díaz, Gustavo Castillo Herrera. “Lutein as a functional food ingredient: Stability and bioavailability.” Journal of Functional Foods 66 (2020): 103771.

https://doi.org/10.1016/j.jff.2019.103771

Sherry, Christina L.,  Jeffery S. Oliver, Lisa M. Renzi, and Barbara J. Marriage. “Lutein supplementation increases breast milk and plasma lutein concentrations in lactating women and infant plasma  concentrations but does not affect other carotenoids.” J. Nutr. 144 (2014): 1256–1263.

http://dx.doi.org/10.3945/jn.114.192914

Stringham, James M., Elizabeth J Johnson, and B Randy Hammond. “Lutein across the lifespan: From childhood cognitive performance to the aging eye and brain.” Curr Dev Nutr 3 (2019): nzz066.

http://dx.doi.org/10.1093/cdn/nzz066

Wang, Wei, Jerome Hernandez, Cecil Moore, Janet Jackson, and Kristina Narfström. “Antioxidant supplementation increases retinal responses and decreases refractive error changes in dogs.” J. Nutr. Sci. 5 e18 (2016): 7 pages

http://dx.doi.org/10.1017/jns.2016.5

Xu, C.Z., H. F. Wang, J. Y. Yang, J. H. Wang, Z. Y. Duan, C. Wang, J. X. Liu , and Y. Lao. “ Effects of feeding lutein on production performance, antioxidative status, and milk quality of high-yielding dairy cows.” J. Dairy Sci. 97;  American Dairy Science Association (2014):7144–7150

http://dx.doi.org/10.3168/jds.2014-8276

Strong demand by consumers; restaurant chains and wholesalers for antibiotic-free (ABF) meat; the threat of antimicrobial resistance; and stringent regulations on the use of antibiotics – there are many good reasons for poultry producers to strive for antibiotic-free production systems. Crucially, to successfully produce poultry meat without antibiotics requires a paradigm shift that starts right at the parent stock level, with the antibiotic-free production of hatching eggs.

Broiler breeders’ gut health is linked to progeny’s performance

Broiler breeders’ performance is measured in terms of how many saleable day old chicks (DOCs) per hen they produce. However, within a sustainable ABF production system (also known as No Antibiotics Ever or NAE), this parameter is not seen in isolation. Breeder hens’ nutritional and health status not only affect the number of DOCs they can produce, but also the transfer of nutrients, antibodies, microbiota and even contaminants, e.g. mycotoxins, to the egg – and therefore, their progeny’s long-term health and performance.

This starts with egg formation, which requires several metabolic processes in the hen to function perfectly. If the hen’s intestinal integrity is compromised, for example due to mycotoxins, she will absorb fewer nutrients, which in turn affects egg formation. Mycotoxicosis has particularly insidious effects for egg formation as it can damage the liver whose biosynthetic activities strongly impact on the egg’s internal (yolk) and external (eggshell) quality.

Chick embryos depend on the maternal antibodies and nutrients deposited in the yolk, including vitamin D3, carotenoids, and fatty acids, to develop normally. Eggshell quality, among other things, affects the embryo’s access to oxygen, which is especially important when it develops body tissues.

Hens’ ability to form healthy eggs depends on their diet and health. Research indicates that, via the impact on egg formation, broiler breeders’ feeding program quantifiably influences their progeny’s immune system and intestinal health. There is indeed a direct relationship between parent and offspring’s gut health because the chick’s microbiome is in part also inherited from the hen. The impact on DOC quality is thus one of many dimensions to consider when calibrating one’s broiler breeders feeding approach.

The challenge of feeding an ABF broiler breeder

Just as their offspring, breeder hens are genetically predisposed for rapid growth and muscle development. From rearing right through to the laying period, poultry nutritionists need to carefully balance their diets and moderate weight gain in order for hens to reach their reproductive potential.

Different stages of a breeder’s life cycle come with different objectives – for example, good flock uniformity in the rearing period versus egg size and hatchability in the laying phase – and thus different requirements in terms of calories, amino acids, vitamins, and minerals. What remains constant is that the actual nutrient intake depends on intestinal health, determining both the breeders’ performance and, via the impact on egg characteristics, its progeny’s performance.

The feeding regimes adopted to avoid hens becoming overweight can have a negative effect on their gut flora. Without antibiotics as a tool to maintain or recover optimal gut function, even mild intestinal disorders can quickly become chronical impairments that negatively impact breeders’ productivity. In ABF production systems, intestinal health therefore needs to be a central focus for the feeding strategy.

Can phytomolecules improve broiler breeders’ performance?

Among the plethora of feed additives, phytomolecules, or secondary plant compounds, stand out as a class of active ingredients that may help to improve gut health and thereby reduce the use of antibiotics.  Synthesized by plants as a defense mechanism against pathogens, phytomolecules combine digestive, antimicrobial and antioxidant properties.

Some studies have shown that phytomolecules-based products can increase broilers’ body weight gain and improve laying hens’ laying rate, egg mass and egg weight. Both broilers and laying hens responded to the inclusion of phytomolecules in their diet with inclusion rate-dependent improvements in feed conversion. To evaluate if phytomolecules could similarly improve broiler breeders’ performance, two trials were conducted.

Study I: Effect of phytomolecules on laying performance during peak production

The first study was set up on a farm in Thailand. In total, 40000 Cobb broiler breeders (85% female, 15% male) were divided into two groups with 8500 hens (one house) in the control and 25500 (three houses) in the trial group. Both groups were fed standard feed. The trial group additionally received a phytomolecules-based liquid complementary feed (Activo® Liquid, EW Nutrition GmbH) via the waterline from week 24 to week 32 at a rate of 200ml/1000L during 5 days per week.

Activo® Liquid was found to have a positive influence on laying performance (Figure 1). The average laying rate increased by 7.2% during the trial period, resulting in almost 3 additional hatching eggs per hen housed. A further indication of the beneficial influence that this particular combination of phytomolecules had on gut health was a 0.2% lower mortality.

Figure 1: Laying rate (%) of breeder hens during first 9 weeks of production

Study II: Effect of phytomolecules on laying performance after peak production

For a second study, conducted in the Czech Republic, 800 female and 80 male Hubbard breeders (JA57 and M77, respectively) were divided into 2 groups with 5 replicate pens and 80 female and 8 male breeders per pen. The experiment started after the peak-production period, at 34 weeks of age and ended at 62 weeks of age. All animals received a standard mash diet. For one group a phytogenic premix (Activo^®, EW Nutrition GmbH) was added to the diet at a rate of 100g/MT.

The results indicate that Activo^® helped maintain the breeder hens’ egg laying performance close to the breed’s genetic potential (Figure 2). In the course of the experiment, Activo® supplemented birds produced 3.6 more eggs than control birds, while consuming a similar amount of feed. As a result, feed consumption per egg produced was lower for birds receiving phytomolecules than for the control birds (169.9 versus 173.6g/d, respectively).

As hatchability was not influenced by the dietary treatment in this study (P>0.5), the 3.6 extra eggs resulted in 2.9 extra day old chicks per hen produced, during the post-peak period alone.
The microencapsulated, selected phytomolecules contained in Activo^® are likely to have improved gut health and feed digestibility, and thereby enhanced the animals’ feed efficiency.

Figure 2: Laying rate (%) of breeder hens week 35 till 62

Chicken or egg? Antibiotic-free poultry production looks at the bigger picture

To successfully produce antibiotic-free poultry meat requires a systematic re-think of each component of the production process. Broiler breeders’ lay the foundation for their progeny’s health and performance via the egg. Breeder hens need to be in optimal health to consistently deliver optimal eggs. Without recourse to antibiotics for maintaining or recovering intestinal functionality, an effective ABF production needs to make gut health central to its feeding approach.

The trials reviewed demonstrate that selected phytomolecules quantifiably boost breeders’ laying performance, increasing the number of hatching eggs and DOCs, while reducing mortality and feed consumption per egg produced. As part of an intelligent antibiotic reduction strategy, the right phytogenic products can be potent tools to help poultry producers achieve their NAE objectives.

by S. Regragui Mazili, T. van Gerwe and M. Caballero

References

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.

Ding, Jinmei, Ronghua Dai, Lingyu Yang, Chuan He, Ke Xu, Shuyun Liu, Wenjing Zhao, et al. “Inheritance and Establishment of Gut Microbiota in Chickens.” Frontiers in Microbiology 8 (October 10, 2017): 1967.

Kuttappan, Vivek A., Eduardo A. Vicuña, Juan D. Latorre, Amanda D. Wolfenden, Guillermo I. Téllez, Billy M. Hargis, and Lisa R. Bielke. “Evaluation of Gastrointestinal Leakage in Multiple Enteric Inflammation Models in Chickens.” Frontiers in Veterinary Science 2 (December 14, 2015): 66.

Moraes, Vera M. B., Edgar O. Oviedo-Rondón, Nadja S. M. Leandro, Michael J. Wineland, Ramon D. Malheiros, and Pamela Eusebio-Balcazar. “Broiler Breeder Trace Mineral Nutrition and Feeding Practices on Embryo Progeny Development.” Avian Biology Research 4, no. 3 (2011): 122–32.

Oviedo-Rondon, Edgar O., Nadja S. M. Leandro, Rizwana Ali, Matthew Koci, Vera M. B. Moraes, and John Brake. “Broiler Breeder Feeding Programs and Trace Minerals on Maternal Antibody Transfer and Broiler Humoral Immune response1.” The Journal of Applied Poultry Research 22, no. 3 (October 1, 2013): 499–510.

Most grains used as feed raw materials are susceptible to mycotoxin contamination. These toxic secondary metabolites are produced by fungi before or after harvest and cause severe economic losses all along agricultural value chains. For livestock, negative consequences include acute effects such as impaired liver and kidney function, vomiting, or anorexia, as well as chronic effects such as immunosuppression, growth retardation, and reproductive problems. Mycotoxin management is, therefore, of the utmost priority for animal producers worldwide.

But how is it that mycotoxins cause such damage in the first place? This article delves into the complex processes that take place when mycotoxins come into contact with the gastrointestinal tract (GIT). The intestinal epithelium is the first tissue to be exposed to mycotoxins, and often at higher concentrations than other tissues. A deeper understanding of how mycotoxins affect the GIT allows us to appreciate the cascading effects on animal health and performance, why such damage already occurs at contamination levels well below official safety thresholds – and what we can do about it.

The intestinal epithelium: the busy triage site for nutrients and harmful substances

When mycotoxins are ingested, they encounter the GIT’s intestinal epithelium (Figure 1). This single layer of cells lining the intestinal lumen serves two conflicting functions: firstly, it must be permeable enough to allow the absorption of nutrients. On the other hand, it constitutes the primary physiological barrier against harmful agents such as viruses, microorganisms, and toxins.

Within the intestinal epithelium, several types of highly specialized cells are involved in epithelial regeneration, nutrient absorption, innate defense, transport of immunoglobulins, and immune surveillance. The selective barrier function is maintained due to the formation of complex networks of proteins that link adjacent cells and seal the intercellular space. Besides, the intestinal epithelium is covered with mucus produced by goblet cells, which isolates its surface, preventing the adhesion of pathogens to the enterocytes (intestinal absorptive cells).

Due to its dual involvement in digestive and immune processes, the intestinal epithelium plays a pivotal role in the animal’s overall health. Importantly, the epithelium is directly exposed to the entire load of ingested mycotoxins. Hence their effects can be problematic even at low concentrations.

Figure 1: The intestinal epithelium

Problematic effects of mycotoxins on the intestinal epithelium

Most mycotoxins are absorbed in the proximal part of the gastrointestinal tract (Table 1). This absorption can be high, as in the case of aflatoxins (~90%), but also very limited, as in the case of fumonisins (<1%); moreover, it depends on the species. Importantly, a significant portion of unabsorbed toxins remains within the lumen of the gastrointestinal tract.

Some of the mycotoxins that enter the intestinal lumen can be bio-transformed into less toxic compounds by the action of certain bacteria. This action, however, predominantly happens in the large intestine – therefore, no detoxification takes place before absorption in the upper parts of the GIT. Part of the absorbed mycotoxins can also re-enter the intestine, reaching the cells from the basolateral side via the bloodstream. Furthermore, they re-enter through enterohepatic circulation (the circulation of substances between the liver and small intestine). Both actions increase the gastrointestinal tract’s overall exposure to the toxins.

Table 1: Rate and absorption sites of different mycotoxins

Adapted from: Biehl et al., 1993; Bouhet & Oswald, 2007; Devreese et al., 2015; Ringot et al., 2006

The damaging impact of mycotoxins on the intestinal epithelium initially occurs through:

• A decrease in protein synthesis, which reduces barrier and immune function (Van de Walle et al., 2010)
• Increased oxidative stress at the cellular level, which leads to lipid peroxidation, affecting cell membranes (Da Silva et al., 2018)
• Changes in gene expression and the production of chemical messengers (cytokines), with effects on the immune system and cellular growth and differentiation (Ghareeb et al., 2015)
• The induction of programmed cell death (apoptosis), affecting the reposition of immune and absorptive cells (Obremski & Poniatowska-Broniek, 2015)

Importantly, studies based on realistic mycotoxin challenges (e.g., Burel et al., 2013) show that the mycotoxin levels necessary to trigger these processes are lower than the levels reported as safe by EFSA, the Food Safety Agency of the European Union. The ultimate consequences range from diminished nutrient absorption to inflammatory responses and pathogenic disorders in the animal (Figure 2).

Figure 2: Mycotoxins’ impact on the GIT and consequences for monogastric animals

1. Alteration of the intestinal barrier‘s morphology and functionality

The mycotoxins DON, fumonisin, and T2 induce a reduction in the rate of epithelial cell proliferation and differentiation. This causes a decrease in the height and the surface of the intestinal villi, which in turn leads to a reduction in nutrient absorption. Additionally, some nutrient transporters are inhibited by the action of mycotoxins such as DON and T2, for example, negatively affecting the transport of glucose.

Several studies indicate that mycotoxins such as aflatoxin B1, DON, fumonisin B1, ochratoxin A, and T2, can increase the permeability of the intestinal epithelium of poultry and swine (e.g. Pinton & Oswald, 2014). This is mostly a consequence of the inhibition of protein synthesis. As a result, there is an increase in the passage of antigens into the bloodstream (e.g., bacteria, viruses, and toxins). This increases the animal’s susceptibility to infectious enteric diseases. Moreover, the damage that mycotoxins cause to the intestinal barrier entails that they are also being absorbed at a higher rate.

2. Impaired immune function in the intestine

The intestine is a very active immune site, where several immuno-regulatory mechanisms simultaneously defend the body from harmful agents. Immune cells are affected by mycotoxins through the initiation of apoptosis, the inhibition or stimulation of cytokines, and the induction of oxidative stress. Studies demonstrate that aflatoxin, DON, fumonisin, T2, and zearalenone interact with the intestinal immune system in such a manner that the animal’s susceptibility to viral and bacterial infections increases (e.g., Burel et al., 2013). Moreover, by increasing their fecal elimination, the horizontal transmission of pathogens is extended.

For poultry production, one of the most severe enteric problems of bacterial origin is necrotic enteritis, which is caused by Clostridium perfringens toxins. Any agent capable of disrupting the gastrointestinal epithelium – e.g. mycotoxins such as DON, T2, and ochratoxin – promotes the development of necrotic enteritis. The inhibition of the intestinal immune system caused by mycotoxins such as aflatoxin, DON, and T2 also collaborates with the development of this disease.

3. Alteration of the intestinal microflora

The gastrointestinal tract is home to a diverse community of bacteria, fungi, protozoa, and viruses, which lines the walls of the distal part of the intestine. This microbiota prevents the growth of pathogenic bacteria through competitive exclusion and the secretion of natural antimicrobial compounds, volatile fatty acids, and organic acids.

Recent studies on the effect of various mycotoxins on the intestinal microbiota show that DON and other trichothecenes favor the colonization of coliform bacteria in pigs. DON and ochratoxin A also induce a greater invasion of Salmonella and their translocation to the bloodstream and vital organs in birds and pigs – even at non-cytotoxic concentrations. It is known that fumonisin B1 may induce changes in the balance of sphingolipids at the cellular level, including for gastrointestinal cells. This facilitates the adhesion of pathogenic bacteria, increases in their populations, and prolongs infections, as has been shown for the case of E. coli.

From the perspective of human health, the colonization of the intestine of food-producing animals by pathogenic strains of E. coli and Salmonella is of particular concern. Mycotoxin exposure may well increase the transmission of these pathogens, posing a risk for human health.

4. Interaction with bacterial toxins

When mycotoxins induce changes in the intestinal microbiota, this can lead to an increase in the endotoxin concentration in the intestinal lumen. Endotoxins or lipopolysaccharides (LPS) are fragments of Gram-negative bacteria’s cell walls. They are released during bacterial cell death, growth, and division. Hence endotoxins are always present in the intestine, even in healthy animals. Endotoxins promote the release of several cytokines that induce an enhanced immune response, causing inflammation, thus reducing feed consumption and animal performance, damage to vital organs, sepsis, and death of the animals in some cases.

The synergy between mycotoxins and endotoxins can result in an overstimulation of the immune system. The interaction between endotoxins and estrogenic agents such as zearalenone, for example, generates chronic inflammation and autoimmune disorders because immune cells have estrogen receptors, which are stimulated by the mycotoxin. The combination of DON at low concentrations and endotoxins in the intestine, on the other hand, has been shown to engender a decrease in transepithelial resistance and to alter the balance of the microbiota.

What to do? Proactive toxin risk management

To prevent the detrimental consequences of mycotoxins on animal health and performance, proactive solutions are needed that support the intestinal epithelium’s digestive and immune functionality and help maintain a balanced microbiome in the GIT. Moreover, it is crucial for any anti-mycotoxin product to feature both anti-mycotoxin and anti-bacterial toxin properties and that it supports the organs most targeted by mycotoxins, e.g., the liver. EW Nutrition’s Mastersorb® Gold premix is based on the synergistic combination of natural clay minerals, yeast cell walls, and phytomolecules. Its efficacy has been extensively tested, including as a means for dealing with E. coli endotoxins.

Mastersorb® Gold: anti-mycotoxin activity stabilizes performance and strengthens liver health

A field trial conducted in Germany on male Ross 308 broilers showed that for broilers receiving a diet contaminated with DON and zearalenone, adding 1kg of Mastersorb® Gold per ton of feed to their diet led to significant performance enhancements. Not only did they recuperate the mycotoxin-induced weight loss (6% increase relative to the group receiving only the challenge), but they gained weight relative to the control group (which received neither the challenge nor Mastersorb® Gold). Feed conversion also improved by 3% relative to the group challenged with mycotoxins.

A scientific study of crossbred female pigs showed that Mastersorb® Gold significantly reduced the deleterious effects of fumonisin contamination in the feed. The decrease in weight gain and the decline of feed conversion could be mitigated by 6.7% and 13 FCR points, respectively (Figure 3). Also, the sphinganine/sphingosine (Sa/So) ratio, a biomarker for fumonisin presence in the blood serum, could be decreased by 22.5%.

Figure 3: Mastersorb® Gold boosts performance for pigs fed a fumonisin-contaminated diet

Another study of crossbred female piglets, carried out at a German university, investigated whether Mastersorb® Gold could support performance as well as liver health under a naturally occurring challenge of ZEA (~ 370ppb) and DON (~ 5000ppb).  Mastersorb® Gold significantly improved weight gain and feed conversion in piglets receiving the mycotoxin-contaminated diet: daily body weight gain was 75g higher than that of a group receiving only the challenge, and the FCR improved by 24% (1.7 vs. 2.25 for the group without Mastersorb® Gold). Moreover, Mastersorb® Gold significantly improved the liver weight (total and relative) and the piglets’ AST levels (aspartate aminotransferase, an enzyme indicating liver damage). A tendency to improve spleen weight and GGT levels (gamma-glutamyl transferase, another enzyme indicative of liver issues) was also evident, all of which indicate that Mastersorb® Gold effectively counteracts the harmful impact of mycotoxin contamination on liver functionality.

In-vitro studies demonstrate Mastersorb® Gold’s effectiveness against mycotoxins as well as bacterial toxins

Animal feed is often contaminated with two or more mycotoxins, making it important for an anti-mycotoxin agent to be effective against a wide range of different mycotoxins. Besides, to prevent mycotoxins damaging the GIT, an effective product should ideally adsorb most mycotoxins in the first part of the animal’s intestine (under acidic conditions). In-vitro experiments at an independent research facility in Brazil showed that an application of 0.2% Mastersorb® Gold binds all tested mycotoxins at rates from 95 to 97% at a pH level of 3, using realistic challenges of 1000ppb (Aflatoxin B1 and ZEA) and 2500ppb (Fumonisin B1 and DON). The binding results achieved for Fumonisin and DON, which are often considered outright “nonbinding,” under challenging close to neutral conditions (pH 6), are particularly encouraging.

Figure 4: Mastersorb® Gold binding capacity against different mycotoxins (%)

Concerning its efficacy against endotoxins, an in vitro study conducted at Utrecht University, among other studies, has shown Mastersorb® Gold to be a strong tool against the LPS released by E. coli. For the test, four premium mycotoxin binders were suspended in a phosphate buffer solution to concentrations of 0.25% and 1%. E. coli LPS were suspended to a final concentration in each sample of 50ng/ml. Against this particularly high challenge, Mastersorb® Gold achieved a binding rate of 75% at an inclusion rate of 1%: clearly outperforming competing products, which at best showed a binding rate of 10%.

Conclusion

A healthy gastrointestinal tract is crucial to animals’ overall health: it ensures that nutrients are optimally absorbed, it provides effective protection against pathogens through its immune function, and it is key to maintaining a well-balanced microflora. Even at levels considered safe by the European Union, mycotoxins can compromise different intestinal functions such as absorption, permeability, immunity, and microbiota balance, resulting in lower productivity and susceptibility to disease.

To safeguard animal performance, it is important to continually strive for low levels of contamination in feed raw materials –  and to stop the unavoidable mycotoxin loads from damaging the intestinal epithelium through the use of an effective anti-mycotoxin agent, which also supports animals against endotoxins and boosts liver function. Research shows that Mastersorb Gold is a powerful tool for proactive producers seeking stronger animal health, welfare, and productivity.

By Marisabel Caballero and Sabria Regragui Mazili

Sub-therapeutic doses of antibiotic growth promoters (AGPs) were used for more than 50 years in poultry production to achieve performance targets – until growing concerns arose regarding antibiotic resistance (Kabir, 2009) and decreasing efficacy of antibiotics for medical purposes (Dibner & Richards, 2005).

Isolates of ESBL-producing E.coli from animals, farmworkers, and the environment were found to have identical multidrug resistance patterns (A. Nuangmek et al., 2018). There is also evidence that AMR strains of microorganisms spread from farm animal to animal workers and beyond. Global AMR fatalities are increasing and might reach 10 million by 2050 (Mulders et al., 2010, Trung et al., 2017, Huijbers et al., 2014).

In light of this, certain AGPs have already been banned, and there is a strong possibility of future restrictions on their use worldwide. Bans are effective: the MARAN report 2018 shows that lower antibiotics usage following the EU ban on AGPs has reduced resistant E.coli in broilers. Another positive consideration is the market opportunities that exist for antibiotic residue-free food.

However, the key element that poultry producers need to get right for antibiotic reduction to be successful is respiratory health management. This article looks at why respiratory health is a particular challenge – and how phytogenic solutions can help.

A closer look at the chickens’ respiratory system

The respiratory tract is equipped with a functional mucociliary apparatus consisting of a protective mucous layer, airway surface liquid layer, and cilia on the surface of the ciliated cells. This apparatus produces mucus, which traps the inhaled particles and pathogens and propels them out of the airways. This mechanism, called the mucociliary clearance, is the primary innate defense mechanism of the respiratory system.

High stocking density combined with stressful environmental factors can negatively influence birds’ immune systems (Heckert et al., 2002; Muniz et al., 2006), making them more susceptible to respiratory disease. When a bird suffers from respiratory disease, which is nowadays usually complicated by a co-infection or secondary bacterial infection, there is an excess production of mucus that results in ciliostasis and, therefore, in an impaired mucociliary clearance. The excess mucus in the tract obstructs the airways by forming plagues and plugs, resulting in dyspnea (hypoxia) and allowing the invasive bacteria to adhere and colonize the respiratory system.

The build-up of mucus in the respiratory tract severely reduces oxygen intake, causing breathlessness, reduced feed intake, and a drop in the birds’ energy levels, which negatively impacts weight gain and egg production. Respiratory problems can result from infection with bacteria, viruses, and fungi, or exposure to allergens. The resultant irritation and inflammation of the respiratory tract leads to sneezing, wheezing, and coughing – and, therefore, the infection rapidly spreads within the flock.

Relatively high stocking density is the norm in poultry production

Low or no antibiotics: how to manage respiratory disease?

Unsurprisingly, respiratory diseases in poultry are a major cause of mortality and economic loss in the poultry industry. For Complicated Chronic Respiratory Disease (CCRD), for instance, although the clinical manifestations are usually slow to develop, Mycoplasma gallisepticum (MG), in combination with E. coli, can cause severe airsacculitis. Beside feed and egg production reduction, these problems are of high economic significance since respiratory tract lesions can cause high morbidity, high mortality, and significant carcass condemnation and downgrading.

Producers need to pre-empt the spread of respiratory pathogens, react quickly to alleviate respiratory distress and maintain the mucociliary apparatus’ functionality. Traditionally, treatment options are based on antiviral, anti-inflammatory, and antibiotic drugs. Can the poultry industry limit losses from respiratory infections without excessive recourse to antibiotics?

Indeed, a sudden reduction in antibiotic usage comes with a risk of impaired performance, increased mortality, and impaired animal health and welfare. The impact has been quantified as a 5% loss in broiler meat production per sq. meter (Gaucher et al., 2015). Effective antibiotics reduction requires a combination of innovative products and suitable consultancy services to manage poultry gut health, nutrition, flock management, biosecurity, and, particularly, respiratory health.

Non-antibiotic alternatives to control diseases and promote broiler growth, such as organic acids (Vieira et al., 2008), probiotics (Mountzouris et al., 2010), prebiotics (Patterson & Burkholder, 2003), and essential oils (Basmacioğlu Malayoğlu et al., 2010) have been the subject of much research in recent years.

Phytogenic solutions: proven efficacy

Essential oils, which are extracted from plant parts, such as flowers, buds, seeds, leaves, twigs, bark, wood, fruits, and roots, have a particularly well-established track record of medicinal applications. Efforts have centered on phytomolecules, the biologically active secondary metabolites that account for the properties of essential oils (Hernández et al., 2004; Jafari et al., 2011).

Studying these properties is challenging: essential oils are very complex natural mixtures of compounds whose chemical compositions and concentrations are variable. For example, the concentrations of the two predominant phytogenic components of thyme essential oils, thymol and carvacrol, have been reported to range from as low as 3% to 60% of the whole essential oil (Lawrence and Reynolds, 1984).

Another well-researched example is eucalyptus oil. The essential oils of eucalyptus species show antibacterial, anti-inflammatory, diaphoretic, antiseptic, analgesic effects (Cimanga et al., 2002) and antioxidant properties (Lee and Shibamoto, 2001; Damjanović Vratnica et al., 2011). The oils are mainly composed of terpenes and terpene derivatives in addition to some other non-terpene components (Edris, 2007). The principal constituent found in eucalyptus is 1,8-cineole (eucalyptol); however, other chemotypes such as α-phellandrene, ρ-cymene, γ-terpinene, ethanone, and spathulenol, among others, have been documented (Akin et al., 2010).

Close-up of eucalyptus leaf oil glands and
the molecular structure of eucalyptol C₁₀H₁₈O (red = oxygen; dark grey = carbon; light grey = hydrogen)

Antimicrobial activity

In modern intensive broiler production, bacterial diseases such as salmonellosis, colibacillosis, mycoplasmosis, or clostridia pose serious problems for the respiratory system and other areas. Analyses of the antibacterial properties of essential oils have been carried out by multiple research units (Ouwehand et al., 2010; Pilau et al., 2011; Solorzano- Santos and Miranda-Novales, 2012; Mahboubi et al., 2013; Nazzaro et al., 2013; Petrova et al., 2013).

Phenols, alcohols, ketones, and aldehydes are clearly associated with antibacterial activity; the exact mechanisms of action, however, are not yet fully understood (Nazzaro et al., 2013). Essential oils’ antimicrobial activity is not attributable to a unique mechanism but instead results from a cascade of reactions involving the entire bacterial cell (Nazzaro et al., 2013). However, it is accepted that antimicrobial activity depends on the lipophilic character of the components.

The components permeate the cell membranes and mitochondria of the microorganisms and inhibit, among others, the membrane-bound electron flow and thus the energy metabolism. This leads to a collapse of the proton pump and draining of the ATP (adenosine triphosphate) pool. High concentrations may also lead to lysis of the cell membranes and denaturation of cytoplasmic proteins (Nazzaro et al., 2013; Gopi et al., 2014).

According to current knowledge, lavender, thyme, and eucalyptus oil, as well as the phytomolecules they contain, show enhanced effects when combined with other essential oils or synthetic antibiotics (Sadlon and Lamson, 2010; Bassole and Juliani, 2012; Sienkiewicz, 2012; de Rapper et al., 2013; Zengin and Baysal, 2014).

Minimum inhibitory concentration (MIC) of some essential oil components against microorganisms in vitro

Immune system boost I: improved production of antibodies

Some essential oils were found to influence the avian immune system positively, since they promote the production of immunoglobulins, enhance the lymphocytic activity, and boost interferon-γ release (Awaad et al., 2010; Faramarzi et al., 2013; Gopi et al., 2014; Krishan and Narang, 2014). Placha et al. (2014) showed that the addition of 0.5g of thyme oil per kg of feed significantly increased IgA levels.

Awaad et al. (2010) experimented on birds vaccinated with the inactivated H5N2 avian influenza vaccine. The experiment revealed that adding eucalyptus and peppermint essential oils to the water at a rate of 0.25 ml per liter resulted in an enhanced cell-mediated and humoral immune response.

Saleh et al. (2014), who applied thyme and ginger oils in quantities of 100mg and 200mg per kg of feed, respectively, observed an improvement in chickens’ immunological blood profile through increased antibody production. Rehman et al. (2013) stated that the use of herbal products containing eucalyptus oil and menthol in broilers showed consistently higher antibody titers against NDV (Newcastle disease virus), compared to untreated broilers.

Immune system boost II: better vaccine responses and anti-inflammatory effects

Essential oils are also used as immunomodulators during periods when birds are exposed to stress, acting protectively and regeneratively. Importantly, the oils alleviate the stress caused by vaccination (Barbour et al., 2011; Faramarzi et al., 2013; Gopi et al., 2014). The study by Kongkathip et al. (2010) confirmed the antiviral activity of turmeric essential oil.

In recent years studies have been carried out on the use of essential oils in conjunction with vaccination programs, including those against infectious bronchitis (IB), Newcastle disease, and Gumboro disease. The results of the experiments show that essential oils promote the production of antibodies, thus enhancing the efficacy of vaccination (Awaad et al., 2010; Barbour et al., 2010; Barbour et al., 2011; Faramarzi et al., 2013).

Essential oils contain compounds that are known to possess strong anti-inflammatory properties, mainly terpenoids, and flavonoids, which suppress the metabolism of inflammatory prostaglandins (Krishan and Narang, 2014). Also, other compounds found in essential oils have anti-inflammatory, pain-relieving, or edema-reducing properties, for example, linalool from lavender oil, or 1,8-cineole, the main component of eucalyptus oil (Peana et al., 2003).

Immune system boost III: antioxidant effects and radical scavenging

An imbalance in the rate of production of free radicals or removal by the antioxidant defense mechanisms leads to a phenomenon referred to as oxidative stress. A mixture of Oregano (carvacrol, cinnamaldehyde, and capsicum oleoresin) was found to beneficially affect the intestinal microflora, absorption, digestion, weight gain and also to have an antioxidant effect on chickens (Bassett, 2000).

Zeng et al. (2015) indicated the positive effect of essential oils on the production of digestive secretions and nutrient absorption. They reduce pathogenic stress in the gut, exert antioxidant properties, and reinforce the animal’s immune status.

Inside the cell, essential oils can serve as powerful scavenger preventing mutations and oxidation (Bakkali et al., 2008). Studies have demonstrated the concentration-dependent free radical scavenging ability of oils from eucalyptus species (Kaur et al., 2010; Marzoug et al., 2011; Olayinka et al., 2012). Some authors attribute the strong antioxidant capacity of essential oils to their phenolic constituents and synergistic effect between tannins, rutin, thymol, and carvacrol, and probably 1, 8-cineole. Moderate DPPH radical scavenging activity reported by Edris(2007), El-Moein et al. (2012), and Kaur et al. (2011).

Vázquez et al. (2012) have demonstrated the potential of the phenolic compounds in eucalyptus bark as a source of antioxidant compounds. The study showed that eucalyptus had ferric reducing antioxidant power in the ranges 0.91 to 2.58 g gallic acid equivalent (GAE) per 100 g oven-dried bark and 4.70 to 11.96 mmol ascorbic acid equivalent (AAE) per 100 g oven-dried bark, respectively (see also Shahwar et al., 2012). Moreover, Eyles et al. (2004) were able to show superoxide dismutase (SOD)-like activity for different compounds and fractions isolated from wood extracts.

Last but not least: positive effects on the respiratory system

In poultry production houses, especially in summer, high temperatures and low humidity increase the amount of air dust. Under such conditions, respiratory tract disorders in broiler chickens, including the deposition of particulates, become more frequent and more severe.

Clinical signs of respiratory disease in chickens include coughing, sneezing, and rales

Thyme oil, thanks to the phytomolecules thymol and carvacrol, supports the treatment of respiratory disorders. These substances smooth tightened muscles and stimulate the respiratory system. An additional advantage lies in their expectorant and spasmolytic properties (Edris, 2007).

These properties are also seen in essential oils such as eucalyptus and peppermint, which contain eucalyptol and menthol. They thin out the mucus and facilitate its removal from the airways. As a result, the airways are cleared and breathing during inflammation becomes easier (Durmic and Blache, 2012).

Another positive effect of the terpenoid compounds used in commercial preparations for poultry is that they disinfect the bronchi, preventing respiratory infections (Awaad et al., 2010; Barbour et al., 2011; Mahboubi et al., 2013). Barbour and Danker (2005) reported that the essential oils of eucalyptus and peppermint improved the homogeneity of immune responses and performance in MG/H9N2-infected broilers.

Grippozon: the phytogenic solution for respiratory health

Grippozon is a liquid composition with a high content of essential oils, which are combined to systematically prevent and ease respiratory diseases. The formulation is derived from the research on essential oils’ effectiveness against respiratory pathogens that are common in animal farming. Grippozon exhibits a synergistic action of all its components to optimally support animal health. It contains a high concentration of active components; both their quantity and quality are guaranteed to deliver results.

Application of Grippozon

Grippozon application can be flexibly adapted to most common housing systems. It is fully water-soluble for use in the drinking line and it is also possible to nebulize a diluted solution in air.

The dose recommendation in drinking water usually amounts to 100ml to 200ml per 1000 liters of drinking water (Grippozon administration has not been reported to affect water consumption). The active substances in Grippozon adhere to mouth mucosa and become volatile in the breathing air later on. Therefore Grippozon can enter the respiratory system indirectly as well. The volatile compounds also spread into the whole barn air and, thus, indirectly via breathing into the respiratory system (and farmers notice the smell of essential oils when Grippozon is applied through in the waterline)

Grippozon can also be used as a spray at a rate of 200ml/10 liters of water for 2000 birds, twice daily on 2-3 days a week. This produces a very effective nebulization effect and offers faster respiratory relief to birds.

Grippozon is an impactful tool for managing respiratory problems. Thanks to its effective mucolytic and relaxant activity, Grippozon gives symptomatic relief to the birds during high-stress periods of respiratory diseases. Mucus in the trachea works as media for the proliferation of bacteria and viruses, so by thinning the mucus, Grippozon slows down the proliferation of bacteria and the spread of disease. Grippozon helps in improving air quality and air intake. It can also be used to stimulate the immune response during vaccination.

Authors:
Ruturaj Patil – Product Manager Phytogenic Liquids
Kowsigaraj Palanisamy – Global Validation Trial Manager

References available on request

Author: Sabria Regragui Mazili, Science Communication Team, EW Nutrition

As people across the globe are re-discovering home cooking, grocery items such as milk, flour, and eggs are in higher demand than ever. Legendary chef Michel Roux once said that “Eggs are more than an element of cooking… The little egg is life.” Let us look at the complex system that allows us to enjoy this superfood – and how egg producers tackle the supply chain disruptions caused by the pandemic.

The coronavirus pandemic has led to increased demand for eggs and, therefore, to temporary shortages

Eggs: from farm to table

The story of our breakfast eggs starts with another set of eggs. The hens and cocks on so-called parent stock or layer breeder farms produce fertilized eggs (1). These “hatching eggs” are transferred to specialized hatcheries where the chicks hatch after 21 days (2). The female chicks, who will go on to become the hens that lay our eggs, are sent to so-called rearing or pullet farms, where they grow up (3). The young hens are then transferred to the layer farms (4).

When they are about 20 weeks old, the hens start to lay – and join the 7 billion chickens that produce more than 1.3 trillion eggs for us every year. During their 14-month laying period, hens will lay 360+ eggs.

Newly laid eggs need to be collected, and checked for quality (grading). About 50-65% of the hens’ output can be sold as “shell eggs,” i.e. whole eggs, to outlets servicing the hospitality industry and end consumers (5). These eggs need to be stamped and then packaged in the egg cartons we all know. “Breaker eggs,” i.e. irregularly sized or broken eggs, do not go to waste: they are moved to processing plants that produce liquid egg and egg powder products for food industry clients such as industrial bakeries and ice cream manufacturers (6).

How many steps it ultimately takes to get eggs from the farm to your plate depends on the complexity of the marketing channel. Farms might do their branding on-site; others work with specialized companies for packaging and branding. Some farms have direct relationships with retailers; others sell to wholesale outlets and distributors. The logistics for getting eggs quickly and safely from A to B involve dedicated egg distribution centers and fleets of refrigerated trucks.

Did you know?

An egg’s color depends on the layer hen’s breed. In the beginning, all eggs are white; those that end up being brown (or, in the case of certain chicken breeds, green or blue) have pigments deposited on them as the eggs pass through the hen’s oviduct.
The reason brown eggs are usually more expensive than white ones is that brown egg layer breeds tend to be heavier and consumer more feed. The eggshell color does not influence the egg’s taste or nutritional value.

Infographic: The egg supply chain, from farm to table

Coronavirus and food: disruptive effects on supply

The impact of the COVID-19 pandemic is felt along each step of this complex process. Disruptions in the supply chain for feed ingredients that mostly come from China, such as amino acids and vitamins, has led to increased price volatility of these ingredients, affecting farmers’ production costs.

Labor availability is a significant concern, for example with respect to the numerous transitions between locations. According to a recent Rabobank report on the situation in Europe, even though food transportation services are exempted from lockdown measures, the high incidence of COVID-19 cases are contributing to driver shortages and higher transport costs. Importantly, everyone working on the “front line” of food supply chains – drivers, as well as farmers, processors, distributors, or retail employees – are doing so at considerable personal risk.

… and effects on demand

Despite these disruptions, animal producers are under pressure to increase production as food consumption patterns are changing in light of COVID-19. In Argentina, for example, the national egg producers association just reported that household consumption of eggs rose by 40% since the country went into lockdown on March 20.

In the US, egg orders from some retailers have increased by up to six times their normal levels, according to market research firm Urner Barry, while wholesale egg prices have risen 180% throughout March. Yet, hens can’t lay more than they already do; to increase flocks takes time and significant investment in additional facilities.

To ease egg supply shortages, the US Food and Drug Administration has temporarily relaxed certain packaging and labeling requirements for shell eggs sold in retail markets. In Canada, egg supplies usually destined for restaurants are being re-directed to meet increased consumer demand.

Egg producers are finding solutions

This would not work in Germany, where, according to egg and poultry analyst Margit Beck, many of the eggs used in the ailing foodservice sector are cage eggs*, for which there is little end consumer demand. Cage egg prices are effectively in free fall, while crisis-induced consumer reflections on purchasing behaviors appear to strengthen demand for free-range and organic eggs. Smaller farmers in Germany and Austria report that they capitalize on this trend through increased direct sales to customers at farm shops and markets.

The sight of (temporarily) empty supermarket shelves might be disconcerting – but, positively, it ought to inspire us to appreciate the incredible work that goes into putting eggs on our tables. Clapping for health care workers has become a new tradition during this pandemic. It would only be fitting to also give a hand for the numerous people along food supply chains whose commitment keeps us all going.

* Note: Conventional battery cages have been banned in Germany since 2010; eggs classified as cage eggs come from laying hens kept in small-group housing systems or in so-called “enriched cages”.

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 Mastersorb 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 Mastersorb 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 Mastersorb Gold reduced the toxin load on the solution by 37%. At 1% inclusion, Mastersorb Gold 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 Mastersorb 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.

The large majority of poultry specialists in Europe consider phytomolecules as one of the key elements in diets for broilers, broiler breeders, and layers when birds are raised without antibiotics. A quick glance at the market will reveal more commercial products than can possibly be imagined. There are three basic elements you should bear in mind when making your choice:

1. Most phytomolecules are volatile. As such, unprotected products will soon evaporate if left exposed to the open air – as it happens, for instance, with feed prepared in commercial farms. Microencapsulation is therefore essential.
2. There are countless phytomolecules. Consequently, finding the right mix for the task required is essential, as not all mixtures will get you the desired result. When designing a phytomolecule mix, the manufacturer must have the necessary knowledge and experience to achieve the desired result.
3. Phytomolecules are powerful. This is to say that you cannot just keep adding higher quantities to achieve a better result. Finding the exact inclusion rates for the right purpose is a difficult balancing exercise.

In fact, the right protection, the right mix and the right inclusion rates must be combined to ensure that the animals do not refuse the feed (worst case scenario) or just fail to benefit from the inclusion of phytomolecules.

Among the feed additives, phytomolecules (or secondary plant compounds) stand out as a class of active ingredients that may help to improve gut health and thereby reduce the use of antibiotics.  Synthesized by plants as a defense mechanism against pathogens, phytomolecules promote the digestion of feed ingredients (Zhai et al. 2018), prevent loss of gut integrity during enteric challenges (Liu et al. 2018), and have antimicrobial properties that hinder the growth of potential pathogens (Chowdhury, 2018). Phytomolecules can prevent the overgrowth of opportunistic pathogens, thereby reducing the frequency of occurrence of diseases such as necrotic enteritis and dysbacteriosis and thus improve performance data such as daily weight gain and feed efficiency.

Beyond the phytomolecules’ proven effects, what works best in supporting the health and welfare of your animals is, in fact, a holistic program (such as those offered by EW Nutrition) that consists of an effective combination of innovative products and consultancy services in the fields of gut health, nutrition, AMR monitoring, and biosecurity management.

*This article is available in Dutch.