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.