Necrotic enteritis (NE) is one of the most common and, at the same time, the most economically devastating diseases of broiler flocks. NE is an infectious disease caused by Clostridium perfringens species, a Gram-positive anaerobic bacterium found in soil, litter, dust and, at low levels, in the healthy gut of birds. C. perfringens causes disease when it converts from a non-producer to a toxin producer; in fact, NE is caused by the species that synthesizes the NetB toxin, which is responsible for the formation of necrotic lesions in the intestine. Although NetB toxin-producing strains of C. perfringens have been confirmed to be the ultimate cause of NE in chickens, it appears that a simple infection is not sufficient to trigger the disease. Predisposing or risk factors are necessary to create a favorable environment for the proliferation of the pathogen.

The classic clinical form of necrotic enteritis is characterized by a sudden increase in mortality of the birds, up to 50%, often without any visible or warning signs, although wet litter can be an indicator of the disease. The subclinical form of the infection is the most frequent: there are no clinical signs of the disease and, in general, not even a mortality peak. Chronic mucosal lesions cause loss of productivity due to poor digestion and absorption, significant weight loss, and increased feed conversion ratio. Although the clinical manifestation of NE can cause high mortality rates, the subclinical form is the most important and harmful since it can persist in the herd without any manifestation. Without symptoms, the chickens are not treated and, as a result, subclinical necrosis causes great economic losses to the poultry industry.

Along with Campylobacter and Salmonella, C. perfringens is one of the most frequently isolated bacterial pathogens in human foodborne illness outbreaks. This confirms that C. perfringens infection in poultry poses a high risk of transmission through the food chain. The use of probiotics could be a valid alternative to prevent the chicken microbiota from being colonized by the pathogen.

Predisposing factors for the development of EN in chickens

Predisposing factors for necrotic enteritis in domestic birds are divided into four main groups (Figure 1): i) changes in the intestine, ii) changes in the immune status of birds, iii) alteration of the microbiota of the gastrointestinal tract (GIT), iv) proliferation of the pathogen C. perfringens.

a) Changes in the intestine. The first precaution that must be taken to prevent C. perfringens infection is to avoid creating an environment favorable for the proliferation of pathogens. Diet thus becomes an important control factor that can influence the appearance and incidence of NE.

Figure 1. The four main factors for the development of necrotic enteritis in chickens.

A diet rich in non-starch and poorly digestible polysaccharides is an important risk factor that predisposes animals to the appearance of NE since, in addition to having a prolonged intestinal transit time, they increase the viscosity of the intestine, creating a favorable environment for the proliferation of C. perfringens. Therefore, wheat, oats, rye, and barley are not recommended for poultry nutrition, as poultry fed these grains are more likely to suffer from NE than those fed corn. Diets rich in non-starch polysaccharides also lead to increased water intake, resulting in wet litter, which can consequently create a favorable environment for the contaminating sporulation of pathogenic bacteria.

Coccidiosis infection is the most well-known predisposing factor for NE. C. perfringens and Eimeria spp. act synergistically to induce typical lesions of necrotic enteritis. Eimeria parasites colonize the small intestine and kill epithelial cells. The physical damage caused by the infection compromises the epithelial integrity of the GIT, and this could lead to serious consequences such as the opening of direct access to the intestinal basal layer, the exposure of extracellular matrix molecules, such as collagen, facilitating adhesion of C. perfringens, as well as the overproduction of mucus that would provide another source of protein-rich nutrients for the proliferation of pathogens.

A high dietary concentration of animal protein, such as fishmeal, has also been found to be a risk factor for the development of NE. C. perfringens lacks many genes required for amino acid biosynthesis, so bacteria cannot grow in an environment where the supply of amino acids is limited. These diets contain poorly digestible proteins that remain in high concentration in the GIT, inducing the growth of C. perfringens and the consequent change in the composition of the microbiota; these effects are modulated by increased nutrients and probably also by increased pH throughout the gastrointestinal tract.

Finally, the physical form of the feed can also influence the incidence of NE: a uniform size of the feed reduces the risk of disease occurrence compared to feed containing particles of heterogeneous size.

b) Changes in immune status. The time of greatest risk for poultry to contract NE is around three weeks of age, when maternal antibodies begin to disappear from the chicken’s bloodstream. These major changes in immune status result in increased susceptibility to C. perfringens infection and proliferation.

Any type of stress can be considered as a potential risk factor that can predispose the animal to the appearance of NE. Overcrowding, environmental ammonia and physiological stress can lower the immune defenses of the chicken, exposing the animal to a possible infection caused by the pathogen. For this reason, a population density that is not too high is recommended, since it could be a predisposing factor for contamination. In addition, exposure to immunosuppressive agents, such as the viruses that cause Marek’s disease, Gumboro disease or chicken anemia, reduces the resistance of animals to GIT infections, increasing the severity of NE.

c) Rupture of the GIT microbiota. Recent research in virology has confirmed that there are no low levels of the population of C. perfringens within the GIT of chickens that under certain predisposing circumstances proliferate to produce the disease, as previously believed, but rather these same strains, which circulate at low levels in healthy birds, they are actually non-pathogenic lines of the same species. Pathogenic strains appear to infiltrate and proliferate in a favorable environment at the expense of nonpathogenic strains to dominate the C. perfringens population and thus induce disease in birds.

Many of the factors that alter the physical state of the gastrointestinal system and the immune status of the animal also affect the composition of the microbiota. It has recently been shown that feeding animals with diets contaminated by Fusarium mycotoxins, deoxynivalenol and fumonisins (FB), could be another predisposing factor for the appearance of necrotic enteritis. It appears that, in addition to coccidiosis, FB-contaminated feed also causes a reduction in the abundance of the segmented filamentous bacteria (SFB) Candidatus savagella, which belongs to a unique group of commensal bacteria within the family Lachnospiraceae. Particularly present in the ileal mucosa of chickens, SFBs play an important role in the modulation of the host’s immune system, especially in the most critical transition period from maternal and innate immunity to endogenous and adaptive immunity. It is precisely in this critical time window that chickens are most likely to contract NE, suggesting the importance of further investigation into the role that SFBs may play in preventing or modulating the disease.

Lactobacillus is one of the predominant genera in the avian gastrointestinal system. These bacteria are important for the role they play in the induction of immunomodulation and for the protection they offer with their antagonistic activities against pathogens. Feeding fishmeal-based diets or FB-contaminated diets has been shown to induce changes in the species composition of lactobacilli within the ceca of chickens, without changing the total count. Lactobacillus johnsonii and Lactobacillus acidophilus suffer a drastic decrease, while the abundance of Lactobacillus reuteri and Lactobacillus animalis increases. The same changes were observed in broilers exposed to the pathogenic strain C. perfringens. Some lactobacilli, such as L. johnsonii, are of great importance for their probiotic activities, including pathogen inhibition. These species ferment carbohydrates introduced through the diet into lactic acid as the main end product, which lowers the intestinal pH and causes inhibition of the growth of acid-sensitive bacteria. Furthermore, the role played by lactobacilli in a cross-feeding process is fundamental: through the production of lactate, they promote the activity of butyrate-producing bacteria.

Butyrate is an important signal molecule of the GIT and also an anti-inflammatory metabolite that participates in the stabilization of intestinal integrity, the improvement of the productive performance of the animal, the change in the composition of the microbiota and the metabolic activity of the entire microbial system in the intestine. The use of fishmeal in the animal diet and the administration of Eimeria have been shown to be two of the main causes of the decrease in the abundance of the populations of Ruminococcaceae and Lachnospiraceae, the main butyrate-producing bacteria in the intestinal system of chickens and mammals. Butyrate has been repeatedly shown to reduce the incidence and severity of necrotic enteritis when given as an additive. Its mode of action is not yet clear, but by colonizing mainly the caeca of chickens, butyrate-producing bacteria can suppress the pathogenic C. perfringens in it, preventing the rise of the infection. Therefore, the signaling function of butyrate makes it an essential metabolite for the universal protective mechanism in all animal species.

d) Proliferation of pathogenic strains of C. perfringens. Nonpathogenic C. perfringens strains are frequently isolated from healthy broilers, whereas only one type of strain predominates in NE-affected animals. It is not yet clear if the chickens that develop the disease already have the pathogenic strain within their GIT that proliferates under favorable conditions, or if certain factors cause the introduction of bacteria into the organism. As explained above, many factors can cause growth, but it is important to understand how selective growth of pathogenic C. perfringens occurs compared to non-pathogenic strains. Alpha toxin was initially thought to be the main virulence factor for NE in broilers, even though both pathogenic and non-pathogenic type A strains produce it. Another study disproved this assumption, showing that the alpha toxin was not related to the lesions caused by the disease. Recently, a new toxin associated with NE in broilers, the NetB toxin, has been discovered. NetB appears to be an efficient environmental adaptation, as it occurs when C. perfringens concentration is high and nutrient availability is limited. The damage that the toxin causes to the host’s cells provides enough nutrients for the bacteria to survive. The discovery that the netB gene encoding the NetB toxin is carried on a conjugative plasmid suggests the possibility of exchanging the plasmid between different strains of C. perfringens and, consequently, the possible transformation of a nonpathogenic strain into a pathogenic one.

Figure 2. The pathogenesis of necrotic enteritis caused by the proliferation of the pathogen C. perfringens and by the production of toxins that destroy intestinal epithelial cells, leads to a blood-tinged diarrhea typical of the disease.

Prophylactic use of probiotics to prevent or mitigate NE

An unstable balance exists between beneficial and non-beneficial bacteria in the normal gastrointestinal tract of healthy chickens. When this balance exists, birds show optimal performance, but under stress conditions, beneficial flora decrease in number, allowing non-beneficial bacteria to grow. This can predispose animals to frequent diseases or even reduce growth parameters and feed efficiency. Probiotics reduce the risk of developing NE by improving the animal’s immunity, the balance of the intestinal microflora, and by stimulating the host’s metabolism. Probiotics, in addition to competing with pathogenic bacteria for available nutrients, also produce microbial substances that inhibit their growth and proliferation.

The composition of the microflora of the GIT of broilers plays a fundamental role in preventing the occurrence of necrotic enteritis. As discussed above, butyrate-producing bacteria can inhibit inflammation of the intestine and preserve its normal activity. Lactic acid-producing bacteria, through a cross-feeding mechanism, promote intestinal colonization by butyrate-producing bacteria. Many other microorganisms have shown the ability to counteract the growth of C. perfringens, including the genera Bacillus, Lactobacillus, Bifidobacterium, Enterococcus, and yeasts.

Figure 3. Beneficial effects of probiotics on the health of broilers.

Bacillus species promote the growth of chickens affected by NE, improving their feed efficiency and facilitating weight gain. In addition to regulating fatty acid synthesis and oxidation-related genes in the liver of diseased chickens, this genus also enhances antioxidant activity. Bacillus spp. produce bacteriocins that inhibit the growth and proliferation of C. perfringens, and their spores are used in feed for their ability to increase the productive performance of chickens and reduce the mortality of birds affected by NE.

The antibacterial properties of lactic acid-producing bacteria allow their use for the prevention of NE. Its probiotic potential is attributed to its ability to produce lactate, bacteriocins, hydrogen peroxide, and enhance host immunity by increasing cytokine expression. There are many species of Lactobacillus that can be used as probiotics, including L. acidophilus, L. animalis, L. fermentum, L. johnsonii, L. mucosae, L. plantarum, L. reuteri, and L. salivarius.

The ability of Enterococcus to produce hydrogen peroxide and bacteriocins with anti-C. perfringens activity, and to inhibit toxin production, makes them a potentially powerful probiotic for the prevention of NE. In chickens exposed to the pathogenic strain, administration of Enterococcus faecium helps alleviate weight loss, intestinal injury, histopathological inflammation, and prevents apoptosis of intestinal cells.

The importance of a founder microbiota in broilers

In commercial practices of poultry industry, newly hatched chicks are exposed to microbes derived from the environment of the hatchery, human handlers, shipping crates, and transport vehicles, before reaching the farm. This process generally takes place within the first few hours of life, at which point there is a rapid increase in bacterial diversity and load in the chicken gut. These first bacterial sources have a significant influence on the development of the intestinal microbiota since it is during these first hours of life when the most significant colonization of the intestine of chickens occurs. Therefore, the absence of the natural environment and contact with adults of the same species makes the founding community of the chick GIT particularly susceptible to human and environmental bacterial sources.

The initial bacterial colonization inhibits or promotes the invasion and stabilization of subsequent bacteria in the intestinal environment and produces metabolites that can support or retard the growth of other bacteria. Therefore, the first days after hatching are critical for effective colonization by beneficial bacteria and selective exclusion of pathogens. The type of diet chickens consume has a major impact on the composition of their gut microbiota, providing nutrients that bacteria can use before or after being processed by the host. Several studies have shown the importance of feeding chicks immediately after hatching, as this increases their productive performance, helps them grow efficiently and drastically decreases mortality.

It follows that, in addition to the immediate administration of essential nutrients for chick growth, a good strategy to ensure the development of an efficient immune system is to administer probiotics that can help form a founder population of commensal bacteria with the ability to selectively exclude pathogenic strains, including C. perfringens. Researchers confirm that the competitive exclusion produced by the inoculation of an adult microflora in day-old chicks has a positive impact on intestinal function and disease resistance. This approach makes it possible to provide the chick with a complete adult microbiota, thus avoiding having to add one or more bacterial strains to an already formed microbiota.

Conclusion and future prospects

Necrotic enteritis caused by Clostridium perfringens bacteria is the cause of huge economic losses in terms of production performance in the poultry industry. Therefore, cost-effective alternatives are urgently sought to remedy frequent NE outbreaks in chicken flocks. A combination of measures aimed at avoiding the predisposing factors and fighting the pathogen seems to be the best strategy to adopt. Probiotics may be the best alternative to replace the use of antimicrobial growth promoters to relieve intestinal inflammation caused by C. perfringens. Many microorganisms have been shown to have effective probiotic activity, including the genera Lactobacillus, Bacillus, Bifidobacterium, and Enterococcus.

More studies are needed to define the colonization mechanism of C. perfringens and to develop new probiotics that can significantly reduce the use of antibiotics to prevent the growth of pathogenic bacteria in the host and thus prevent the occurrence of necrotic enteritis.

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