Etiology and pathogenesis

C. perfringens type A almost exclusively produces clostridial necrotic enteritis of poultry, although it is thought that type C may be involved on rare occasions. However, since most diagnoses of necrotic enteritis are based on gross, and occasionally microscopic, pathology alone, it is possible that type C is involved in at least some cases, but goes undetected.

The disease starts when C. perfringens multiplies anarchically in the intestine of chickens, producing toxins that cause necrosis. In fact, during initial disease development, a single C. perfringens clone arises to dominance in the bird’s intestinal tract. After birds recover from the disease, whether naturally or via antimicrobial treatment, a diverse population of C. perfringens isolates returns. Furthermore, a high portion of NE isolates inhibits the growth of other C. perfringens strains, particularly compared to isolates from healthy birds. Recently, perfrin, a novel bacteriocin, was identified from an NE isolate, and is predominantly associated with NE isolates. This may be a critical virulence factor that allows the NE isolate to arise to dominance, fill open niches, multiply, and establish disease in the host. NE isolates adhere to extracellular matrix molecules, such as fibrinogen and collagen types III and IV, more efficiently than isolates from healthy birds. This suggests a role for some unknown attachment factor in initial colonization. Additionally, proteolytic enzymes affecting the basement membrane of the villous enterocytes help to establish the initial infection by these NE isolates. Overall, research is slowly deciphering the virulence factors needed to establish disease in the host.

For many years, alpha toxin (CPA, the only so-called “major” toxin produced by C. perfringens type A) has been considered the major virulence factor involved in necrotic enteritis, because crude preparations of this toxin obtained from C. perfringens type A cultures produced lesions typical of necrotic enteritis when injected into the intestine of conventional chickens. When these crude toxin preparations were neutralized with anti-CPA serum, no lesions were observed. In addition, CPA has been found in the feces and intestinal content of poultry with NE, and many NE isolates were found to produce higher levels of CPA in vitro than C. perfringens isolates from healthy birds. Thus, it was initially speculated that CPA was the major virulence factor responsible for NE. This was also supported by the fact that vaccination of chickens with recombinant CPA or CPA toxoids provides a degree of protection against NE.

Despite the evidence presented above, during the past few years, the role of CPA in the pathogenesis of NE has been questioned. Fulfillment of molecular Koch’s postulates showed that a CPA null mutant produced lesions typical of NE. Based on that, researchers concluded that CPA was not critical for the pathogenesis of NE. However, additional work has found that CPA is still present in the intestinal tract of birds challenged with a CPA null mutant, as the type A normal flora in the bird’s intestinal tract produces CPA in these circumstances. Additionally, spontaneously derived CPA mutants of NE strains lost the ability to produce disease; however, these mutants were never complemented for CPA production and then tested in vivo. Also, CPA vaccination research has shown that some anti-CPA antibodies bind to the cell wall of C. perfringens and prevent growth, thus potentially providing protection by binding directly to the bacteria rather than to the actual toxin. Therefore, additional work on the importance of CPA in the pathogenesis of NE is needed.

Recently, a toxin called necrotic enteritis toxin (NetB) was discovered and determined to have a critical role in the pathogenesis of NE. NetB shares 38% identity with C. perfringens beta toxin (CPB) and 31% with Staphylococcus aureus alpha hemolysin, both of which are pore-formers. NetB also forms heptameric pores; it also has enhanced activity in the presence of cholesterol and is toxic for chicken hepatocytes (LMH cells). Importantly, a C. perfringens netB null mutant strain did not produce NE lesions in chickens, and this mutant recovered full virulence when netB was restored. The netB gene is found on a large, conjugative plasmid within a 42 kb pathogenicity locus. It is regulated by the VirSR two-component signal transduction system, which is activated as a result of population density and regulates production of a number of other toxins in C. perfringens. Interestingly, IgY levels against both CPA and NetB are significantly higher in healthy chickens compared to birds with NE, which further supports the role of these two toxins in the pathogenesis of NE no matter how minor the effect may be on the disease.

Additional work does need to decipher the exact role of NetB in producing disease, because although some studies have found that 70–90% of NE isolates have netB, and very few C. perfringens isolates from normal chickens have this gene, others have found an almost equal distribution of netB between NE isolates and poultry normal flora isolates. Furthermore, the gene is very highly conserved, but less than 30% of netB-positive isolates from healthy birds actually produce the toxin, while over 90% of netB-positive isolates from NE cases produce the toxin. In addition, a study also reported a netB-negative NE isolate that produced disease in an experimental model. Furthermore, during studies with the netB mutant, the authors found a small number of NE lesions in birds challenged with the netB mutant, but concluded these were due to netB-negative normal flora strains. Conversely, other studies have found that netB-negative NE isolates do not produce disease, and all netB-positive isolates, regardless of the source, produce NE, but with significant differences in severity and incidence.

Beta2 toxin (CPB2) and C. perfringens enterotoxin (CPE), two toxins produced by some C. perfringens isolates, have also been suggested to play a role in poultry NE. However, there is no definitive evidence that either of these toxins plays a role in the pathogenesis of poultry NE. Both are uncommon finds in the genotyping process.

TpeL, a member of the large clostridial toxins (LCT), is present in strains that produce severe disease in experimental NE models. TpeL has been found in less than 10% of isolates from NE birds and is always associated with NetB; however, no isolates from healthy birds have been found to carry tpeL. Although there is no doubt of the role played by NetB in the pathogenesis of many cases of NE, further investigation is needed to determine the possible role of other toxins in some cases of this disease.

Several unknown virulence factors appear to have a potential role in the pathogenesis of NE, and additional approaches, such as comparative genomics, are helping to identify these factors. Comparative genomics of seven NE strains has revealed three highly conserved NE-associated loci including NELoc-1 (42 kb, encoding netB, two leukocidins, and 34 additional genes), NELoc-2 (11.2 kb), and NELoc-3 (5.6 kb). NELoc-1 and NELoc-3 are both located on large, distinct plasmids; in fact, netB-positive isolates have been found always to contain one to four large plasmids. Comparative genomic hybridization of 54 C. perfringens isolates from NE or healthy birds revealed 142 genomic regions variably present in poultry isolates with 49 significantly associated with NetB, while multilocus sequence typing (MLST) studies have identified two major NE clonal groups, and further correlated NetB with NE disease. Overall, this suggests type A strains acquire the plasmid carrying netB, and then through two different pathways acquire other virulence factors to emerge as NE strains.

Host response

Although little is known about the avian immune response to NE, some work has begun to shed light on the issue. Chicken immune microarrays demonstrated that both cell-mediated and humoral-mediated immune responses via MHC classes I and II were activated during NE. While toll-like receptor-2 (TLR-2) appears to be strongly involved in the immune response, several other toll-like receptors, such as TLR-4 and TLR-7, have a minor role in the immune response. A number of cytokines such as TNF-α, IL-8, IL-6, and IL-1β are all upregulated during NE. Mucin has a critical role in protecting the intestinal epithelium during infections. The presence of C. perfringens during NE results in the downregulation of both MUC2 (widely expressed in goblet cells from the small and large intestines) and MUC13 (the exact role is not known) genes in birds, and is suggested most likely to be the consequence of severe shedding of the jejunal mucosa during the disease. The presence of C. perfringens and the development of NE, as expected, also causes major intestinal microflora changes. In particular, various Weissella species and lactobacilli are suppressed by the presence of C. perfringens during disease development. Even though there is limited knowledge about the host’s response to NE, we are beginning to gain a better understanding that will hopefully assist in the development of effective prevention methods.

Predisposing factors

A number of factors have been linked to increasing rates of NE in birds, particularly broiler chickens. The normal number of C. perfringens is negligible in the crop and steadily increases to the highest levels in the colon. Normal, healthy chickens typically have 10²–10⁴ colony-forming units (CFUs)/g of intestinal contents in the small intestine, while birds suffering from NE reach levels of 10⁷–10⁹ CFU/g, demonstrating that predisposing factors which allow high proliferation of C. perfringens are critical for disease development. A preceding or co-infection with Eimeria is known as a major precursor for NE in chickens. Damage caused by Eimeria results in the leakage of plasma proteins into the intestinal tract, which enhances C. perfringens growth and toxin production. High energy, high protein, and/or high non-starch-polysaccharide-containing diets induce higher rates of NE in birds. An increased level of animal protein such as fishmeal in feed has been a known predisposing factor for years, as animal protein is poorly digested, providing large amounts of cysteine, glycine, and proline to C. perfringens for growth and toxin production. Recently, inclusion of potato protein in the diet resulted in higher rates and more severe NE than either fishmeal or soybean diets. Additionally, potato protein contains higher trypsin inhibitor activity than fishmeal or soybean; trypsin destroys C. perfringens toxins, so inhibitors have been associated with a higher incidence of NE in birds. In addition to the physical damage and providing critical nutrients to C. perfringens, examination of the microbiota has revealed that fishmeal and Eimeria infection both cause a significant change in the alpha and/or beta diversity of the intestinal tract of broiler chickens which allows C. perfringens to colonize and expand in numbers. High non-starch polysaccharides, such as from wheat-, rye-, or barley-based diets, increase digesta viscosity, which slows intestinal movement, increases mucus production, and impairs oxygen transfer, allowing C. perfringens to grow. Additionally, the size of the feed particles can increase the incidence of NE, as finely ground feed provides more surface area for C. perfringens to metabolize the feed and proliferate faster.

Interestingly, the common feed contaminant Fusarium mycotoxin deoxynivalenol also has the potential to more than double the occurrence of subclinical NE, due to the intestinal epithelial damage the mycotoxin produces, which increases nutrient availability to C. perfringens. Aflatoxin B₁ also results in increased occurrence, severity, and mortality rates due to NE, which has been suggested to be due to suppression of the bird’s immune system by the toxin. In addition to diet, higher stocking densities of broiler chickens increase the severity of the NE lesions. Although these predisposing factors can make birds more susceptible to disease, research has proven that a virulent C. perfringens NE isolate must be present under these conditions in order for disease to develop.

Clinical signs

Acute and subclinical forms of the disease have been described. Chickens with acute NE show a variety of clinical signs, which may include depression, reluctance to move, diarrhea, ruffled feathers, decreased appetite or anorexia, huddling, dribbling from the beak, and dehydration. The course of the acute disease is usually very short and most birds die within 1–2 hrs after the onset of clinical disease; finding birds dead without premonitory clinical signs is not uncommon. An early indicator of disease can be wet litter, although this can be coincident with, rather than prior to, disease development. Mortality rates due to acute NE can reach as high as 50%.

Chickens with the subclinical form of NE suffer a drop in production, but no other clinical signs are evident. The chronic damage to the intestinal mucosa results in reduced weight gain and higher feed conversion ratios due to decreased digestion. Clinical signs of NE in birds of other species are similar to those described in chickens, while subclinical disease has only been described in chickens.