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Necrotic enteritis: Applications for the poultry industry
©2014 Poultry Science Association, Inc.
Necrotic enteritis: Applications for the poultry industry Diego Paiva and Audrey McElroy1
Primary Audience: Researchers, Veterinarians, Nutritionists, Live Production Managers SUMMARY Considering market demands concerning the decreased use of growth promoters and anticoccidial drugs in feed formulations, the poultry industry has been trying to reduce or eliminate the inclusion of subtherapeutic doses of antimicrobials into feed. Formulating diets not only to meet birds’ nutrient requirements for growth but also for gastrointestinal health parameters is increasingly important. Maintenance and enhancement of intestinal integrity is essential for bird performance when antimicrobials are not included in feed, as commercial poultry face numerous enteric pathogen challenges. Necrotic enteritis has reemerged as an important disease of poultry in recent years. The reduction in the use of antimicrobials in poultry feeds has been attributed as one of the main contributing factors for the increasing incidence of necrotic enteritis (NE) in commercial poultry. Mortality due to NE is extremely high (1% daily mortality), which results in great economic losses. Economic losses due to NE are not only associated with high mortality, but also associated with decreases in bird performance and FE, particularly in subclinical cases of NE. Birds that survive NE outbreaks usually have a reduced ability to digest and absorb nutrients due to extensive damage to the mucosal lining, which ultimately results in reduced profitability. Key words: necrotic enteritis, Clostridium perfringens, netB, alpha-toxin, chicken, enteritis 2014 J. Appl. Poult. Res. 23:557–566 http://dx.doi.org/10.3382/japr.2013-00925
INTRODUCTION Necrotic enteritis (NE) is an enterotoxemia of poultry with an important economic effect on poultry production. It has been estimated that NE costs the poultry industry $2 billion globally as a result of reduction in bird performance and disease treatment [1, 2]. The reduction in bird performance is not only associated with impaired growth rate and feed conversion during production, but also with increased condemna-
1
Corresponding author: [email protected]
tion rates in broilers due to hepatitis at the processing plant [3, 4]. In the past, the use of antimicrobial growth promoters (AGP) in commercial poultry feed helped control NE in poultry flocks [5, 6]. However, NE has reemerged as a significant problem in poultry production, likely as a result of national and international policies that ban or limit the use of AGP in poultry feeds [4, 5, 7]. In addition, consumers’ preferences have had a large effect on bird production, and the push for poultry
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Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061
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558 production with less medication in the diet has also had a significant effect on the increased incidence of NE in the past few years [7]. The objective of the current review is to discuss recent findings in the pathogenesis of NE and alternative prevention and treatment options.
ETIOLOGY AND EPIDEMIOLOGY
PREDISPOSING FACTORS Clostridium perfringens is a naturally occurring bacterium in the intestines of warmblooded animals, and its presence alone is not a determining factor for disease development. Therefore, predisposing factors that could lead to an overgrowth of C. perfringens are crucial to NE onset and development. Several predisposing factors for NE have been identified, including dietary factors, immune status and stress, intestinal physiopathology, and coccidiosis. Dietary Factors The gastrointestinal tract ecosystem is very susceptible to diet composition. Different inclusion levels of specific nutrients can have a significant effect on the microbial population in the intestine and ultimately affect bird performance and susceptibility to diseases. Several different dietary components may favor C. perfringens growth and consequently NE onset and development. The type of cereal grain used in poultry diets has been identified as a dietary component affecting the onset of NE. Research has shown that the use of diets formulated with cereals (barley, rye, oats, and wheat) containing high levels of indigestible, water-soluble, nonstarch polysaccharides (NSP) predisposes birds to NE [17, 18]. Whereas the mechanism by which NSP predisposes birds to NE is not clear, the effects that NSP have on bird performance, nutrient digestibility, and digesta viscosity have been extensively reported. Complex carbohydrates are known to have a negative effect on bird performance [19, 20]. The reduction in performance can be associated with a decrease in nutrient digestibility of several nutrients, such as amino acids, fat, and cholesterol, as well as decreased DM retention [20–22]. These nutrients then
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Clostridium perfringens (types A and C) is the etiological agent associated with NE [4]. Clostridium perfringens is a gram-positive, rodshaped, anaerobic encapsulated bacterium that causes a broad spectrum of human and veterinary diseases [8–10]. Clostridium perfringens differs from many other clostridia in being nonmotile, reducing nitrate, and carrying out stormy fermentation of lactose in milk [10, 11]. Clostridium perfringens also ferments glucose, fructose, galactose, inositol, maltose, mannose, starch, and sucrose. The fermentation products include acetic and butyric acids with or without butanol [8]. Clostridium perfringens will grow over a wide pH range, varying from 5.5 to 8.5, whereas optimum growth of this bacterium occurs at pH 6 to 7 [9, 11]. The virulence of C. perfringens largely results from its prolific toxinproducing ability. The classification scheme for C. perfringens assigns isolates to 1 of the 5 types (A–E) depending upon their ability to express 1 or all 4 “typing” toxins (α, β, ε, and ι) [8–10]. Clostridium perfringens has a wide distribution, being isolated from the intestine of warmblooded animals and a variety of environments, such as soil, water, and feed [3, 8]. Colonization in the intestines of poultry may happen as early as the day of hatch in the hatcheries. It has been reported that 105 cfu/g of C. perfringens in ileal digesta is the threshold for the development of NE in chickens [12]. Craven et al. [13] were able to isolate C. perfringens from eggshell fragments, chicken fluff, and paper pads in commercial broiler hatcheries. Given that C. perfringens is a naturally occurring bacterium in the intestinal environment of poultry, disease development is dependent on other predisposing factors that will be discussed further in the current review. Although chickens from 2 wk to 6 mo of age are susceptible to NE, incidence varies greatly according to type of bird and rearing conditions.
Most NE outbreaks in broiler flocks raised on litter are reported between the second and fifth week of age [4, 5, 14]. In contrast, NE outbreaks in commercial layers raised in floor pen settings have been reported between 3 to 6 mo of age [4]. Mortality rates associated to NE in broilers are often between 2 and 10%; however, rates as high as 50% have been reported [15, 16].
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Mineral levels in the diet have also been identified as dietary factors that could predispose birds to NE. Zinc was the first mineral associated with NE through its involvement α-toxin activity. The α-toxin produced by C. perfringens depends on zinc to hydrolyze membrane lipids [15, 29, 30]. This mechanism is discussed in detail in the C. perfringens toxins section of the current review. Dietary levels of Ca have also been identified as a relevant nutrient for the pathogenesis of NE, but the mechanism by which Ca favors C. perfringens is unknown. It has been suggested that excess Ca may increase intestinal pH [31, 32] and favor C. perfringens growth in a more neutral environment [5, 33]. Paiva et al. [34] reported that mortality due to NE was significantly higher when feeding broilers 0.9% Ca in the diet when compared with birds fed 0.6% Ca in the diet. However, differences in gastrointestinal pH were not observed in their study when feeding 2 different levels of Ca in the diet during naturally occurring NE, suggesting that Ca was required for α-toxin and netB activity through mechanisms that will be further discussed in the present review. Immune Status and Stress Immunosuppression predisposes animals to NE, because the factors that usually lead to immunosupression likely alter the intestinal environment and the intestinal microbial population [14, 15]. Pathogens that lead to immunosuppression (Eimeria spp., infectious bursal disease virus, chick anemia virus, and Marek’s disease virus) have been reported to predispose chickens to NE [2, 16]. In an NE disease model study, McReynolds et al. [2] reported that birds infected with infectious bursal disease virus often have secondary infections with C. perfringens. Stress likely has the same immunosuppressive effects of the aforementioned pathogens. Environmental (heat or cold stress) and managerial (feed changes, litter conditions, stocking density, vaccination programs) stressors have been shown to cause immunosupression, predisposing birds to disease [2, 15, 35]. Intestinal Physiopathology When intestinal conditions are not favorable, even highly virulent C. perfringens strains fail
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become available to microbial utilization in the lower small intestine and could potentially change the type and balance of intestinal microbiota [22, 23]. Jia et al. [24] reported that digesta was more viscous in birds consuming diets containing high levels of NSP. The increase in digesta viscosity due to NSP has been associated with an increase in mucous secretion [23] and an increase in water-binding capacity by complex carbohydrates [19, 22]. High digesta viscosity impairs nutrient digestion by interfering with the interaction between digestive enzymes and their substrates and impeding nutrient uptake in the gastrointestinal tract [19, 20, 22]. As a result, an influx of undigested nutrients enters the distal segments of the small intestine increasing microbial growth. The increased mucous secretion results in a nutrient rich environment favoring C. perfringens over other bacterial species [23], as this bacterium is known to have mucolytic activity and a short generation time [25–27]. Dietary protein levels and protein source are also factors that can predispose birds to the development of NE. Protein-rich diets result in a high concentration of protein in the gastrointestinal tract that will serve as substrate for microbial growth [14]. In addition, in the small intestine, proteins are degraded to nitrogenous compounds (ammonia and amines) that not only increase intestinal pH (high logarithmic acid dissociation constant of nitrogenous compounds), but also favor the proliferation of pathogenic bacteria, including C. perfringens [8, 15]. Evidence suggests that protein source may be more important than the protein levels in the diet for NE development. High inclusion levels of animal protein sources (fishmeal, meat and bone meal) are usually associated with an increase in the incidence of NE in broilers [28]. Timbermont et al. [14] hypothesized that animal protein sources would have higher levels of indigestible protein that would reach the ceca and serve as substrate for C. perfringens. Another possibility is that the amino acid balance (especially methionine and glycine) in these feed ingredients would ultimately favor C. perfringens growth in the ceca, leading to NE [5, 28]. However, it is still unclear why high inclusion levels of animal protein in the diet of broilers results in an increase in the ocurrence of NE.
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Coccidiosis Coccidiosis is among the most studied predisposing factors of NE. Coccidiosis in chickens is usually caused by the association of 2 or more species of Eimeria: Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox, and Eimeria tenella. Coccidia oocysts are ubiquitous in areas where poultry are raised, allowing this parasite to cycle and remain in the environment. The Eimeria spp. life cycle is complex and involves stages inside and outside of the host. The consequence of the Eimeria cycle is extensive damage to the intestinal mucosa. The intestinal lumen then becomes rich with plasma proteins, which are themselves rich in amino acids, growth factors, and vitamins that serve as substrate for clostridial growth [3, 5, 14]. In addition, digestibility of nutrients is reduced due to extensive gastrointestinal damage, which may substantially increase nutrient availability for C. perfringens [5, 37]. Furthermore, coccidiosis induces
a local T cell-mediated inflammatory response that increases mucin (and mucous) production [27, 37], which ultimately favors C. perfringens growth due to its mucolytic ability [26, 27]. Clostridium perfringens Toxins Clostridium perfringens is an extracellular pathogen, thus its virulence is mostly associated with its ability to produce toxins. Clostridium perfringens can produce up to 17 toxic or potentially toxic exoproteins [38]. The species is divided into types (A, B, C, D, and E) according to the production of 4 major toxins: α, β, ε, and ι. Type A comprises the strains that produce α-toxin, type B as strains that produce α, β, and ε toxins, type C as strains that produce α and β toxins, type D as strains that produce α and ε toxins, and type E as strains that produce α and ι toxins [38, 39]. As only C. perfringens types A and C are associated with NE, only α and netB toxins will be discussed in the present review. Alpha-Toxin Alpha-toxin is a zinc-dependent phospholipase sphingomyelinase C and is encoded by the phospholipase C gene [7, 29, 30]. Alpha-toxin is organized in 2 distinct domains: the N-terminal region (247 residues), which carries the active site required for phospholipid hydrolysis, and the C-terminal region (123 residues), which carries the lipid-binding site [29, 39]. Alpha-toxin requires zinc for substrate hydrolysis [15, 29, 30], and Ca ions are essential for the binding of α-toxin to lipid films [30, 39, 40]. High Ca concentrations are required for optimal enzymatic activity with physiological substrates [29, 41]. Alpha-toxin has been shown to be cytotoxic, leading to cell lysis of erythrocytes, phagocytes, fibroblasts, platelets, leukocytes, endothelial cells, and myocytes [29, 38]. Alpha-toxin hydrolyses phospholipids and promotes cellular membrane disorganization. In vivo, the cellular substrates for α-toxin are phosphatidylcholine and sphingonyelin, which are both components of the cellular membrane of epithelial cells in the gastrointestinal system [15, 30]. The mechanism of membrane recognition is a complex event involving Ca-mediated phospholipid recognition, where Ca ions are
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to produce disease. However, some intestinal physiopathological circumstances exist that favor the development of NE, such as intestinal stasis, changes in gastrointestinal pH, and damage to the intestinal mucosa [5, 7, 16]. When intestinal motility is reduced (i.e., increased digesta viscosity) feed passage is delayed. This increases nutrient availability to the microbial population in the gastrointestinal tract, allowing C. perfringens to outgrow other species [5]. Associated with an increase in nutrient availability, an increase in transit time also means a reduction in microbial flushing from the gastrointestinal tract, which increases the opportunity of pathogens to proliferate, colonize, and cause disease. Intestinal acidity can also have an effect on NE onset. Higher intestinal pH can predispose birds to develop NE, as C. perfringens growth is inhibited in more acidic intestines [36]. Damage of the intestinal mucosa is another condition that predisposes broilers to develop NE. This predisposition is likely a combination of effects, such as changes in intestinal pH, excess nutrients in the gastrointestinal tract, leakage of plasma proteins and growth factors to the intestinal environment, and loss of intestinal integrity.
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not the main virulence factor of NE. One of the hallmarks of NE in broilers is granulocyte migration from the tissue to the intestinal lumen [47]. This inflammatory reaction is very different from the leukostasis and lack of inflammatory response induced by α-toxin in gas gangrene [48], indicating that NE lesions are probably mediated by toxins other than α-toxin. NetB Toxin NetB, another toxin associated with NE occurrence in broilers, is a pore-forming toxin, with similarity to C. perfringens β-toxin (38% identity), C. perfringens gamma-toxin (40% identity), Staphylococcus aureus α-hemolysin (30% identity), and Staph. aureus gamma-toxin (23% identity) [49, 50]. NetB forms discrete pores of at least 1.6 nm in diameter in the cellular membrane, causing an influx of ions (Ca, Na, Cl, and so on) that eventually lead to osmotic cell lysis [49, 50]. The entrances of the pores formed by netB are negatively charged, which explains why netB pores are cation-selective [50, 51]. NetB is secreted as a water-soluble monomer that is thought to oligomerize on the target cell surface before forming pores [52]. Although Savva et al. [50] reported that netB is able to oligomerize and form pores in the absence of cholesterol, the conversion to oligomer and pore formation is significantly increased in the presence of cholesterol. It was initially suggested that birds were more susceptible to NE than mammals due to differences in cholesterol concentration in the cell membrane of birds. Yan et al. [51] reported differences in susceptibility of avian and mammalian red blood cells to netB mediated lysis. However, as avian and mammalian red blood cells have similar proportions of cell membrane cholesterol, these differences seem to be unrelated to cholesterol levels. The exact role of membrane cholesterol in netB function is still unclear. In addition to promoting cell lysis through an osmotic imbalance, the Ca influx caused by netB may also lead to a special type of programmed cell death. Kennedy et al. [53] reported that the pore-forming α-toxin of Clostridium septicum forms Ca-permeable pores, which increase intracellular Ca. What was surprising about their findings was the fact that the Ca influx did not
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partially coordinated by acidic amino acid side chains of α-toxin and partially by phosphate groups or membrane phospholipids [30]. Hydrolysis of cell membrane phospholipids results in the formation of diacylglycerol, following an activation of protein kinase C and consequent activation of arachidonic acid cascade [15, 39, 41]. The outcome of the activation of the arachidonic acid cascade is the synthesis of inflammatory mediators (leukotrienes, tromboxane, platelet-agglutinating factor, and prostacyclin), which cause blood vessel contraction, platelet aggregation, and myocardial dysfunction, leading to acute death [3, 39, 41]. Alpha-toxin has been indicated as the main virulence mediator for NE in poultry. Al-Sheikhly and Truscott [42] were able to successfully reproduce NE in birds infused intraduodenally with bacteria-free crude toxin. Another indication of α-toxin participation in NE pathogenesis is that C. perfringens isolates from broilers with NE produce significantly more α-toxin than isolates from broilers without NE [43]. Studies of broiler immune response to NE also suggest that α-toxin is an important virulence factor in the pathogenesis of NE. Broilers with NE have significantly lower antibody levels against α-toxin than healthy controls [6, 44]. Higher levels of toxin-reactive antibodies in healthy chickens, when compared with chickens with NE symptoms, indicate that these antibodies may protect birds from developing NE. Despite these reports, the role of α-toxin as the main virulence factor for producing NE in broilers is a very controversial topic. Interpretation of early results can be disputed because most of the studies that reported α-toxin as the main virulence factor in the pathogenesis of NE used crude supernatant instead of purified α-toxin [42, 43]. Conclusions of these studies were based on α-toxin being the most dominant protein present in crude supernatants and did not consider the presence of other toxins in crude supernatants that might be collaborating or even responsible for the development of NE [45]. The most convincing evidence that α-toxin is not the main virulence mediator in the development of NE comes from Keyburn et al. [46], where an α-toxin-negative mutant of a C. perfringens strain from an NE outbreak was still able to produce NE in broilers. Other factors of NE pathogenesis also indicate that α-toxin is
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CLINICAL SIGNS AND PATHOLOGY Necrotic enteritis clinical signs are common to enteritis in general: depression, anorexia, diarrhea, dehydration, and ruffled feathers [3, 4, 7]. The classic acute form of NE is characterized by a sudden increase in flock mortality without any warning clinical signs [3, 14]. The subclinical form of NE is usually mild with no clinical signs or peak in mortality; most of the time, only an overall reduction in bird performance is observed [3, 43]. Performance losses are associated with chronic intestinal mucosal damage resulting in poor nutrient digestion and absorption, reduced BW gain, and increased FCR [3, 14]. In cases of subclinical NE, an increase in
liver condemnations at the processing plant is often observed due to cholangiohepatitis [3, 14]. Therefore, the subclinical form of NE is harder to diagnose and birds are not treated, resulting in greater economic losses [14]. Macroscopic lesions are usually restricted to the jejunum and ileum, but may extend to the duodenum and ceca [4, 7, 14]. The small intestine is usually distended and filled with gas; intestinal walls are thin and friable [4, 14]. Necrosis is visible in the intestinal mucosa, along with presence of a green to yellow diphtheritic membrane that is adherent to the mucosa [4]. Histopathology reveals a severe inflammatory response to C. perfringens. Inflammatory infiltrate is characterized by the presence of heterophils, lymphocytes, macrophages, and plasma cells [14, 47]. Diffuse and severe coagulative necrosis of the mucosa is also observed [14, 47]. Masses of tissue fragments, necrotic cells, cell debris, and bacterial colonies comprise the diphteric membrane characteristic of NE [14, 47]. Blood vessel congestion can be observed in the lamina propria and submucosa.
PREVENTION AND TREATMENT Necrotic enteritis prevention is usually associated with management practices that minimize the effects of the predisposing factors that contribute to disease development. Reducing the inclusion of dietary ingredients that may lead to NE, such as fish meal, oats, barley, and rye, has been a noteworthy solution in decreasing NE incidence [7]. The use of AGP in feed has also played an important role in the control of NE. The introduction of AGP in the diet assists with coccidiosis management and modifies the intestinal microbial populations, which both result in a reduction in the incidence of NE [3, 7]. However, government bans, pathogen resistance to antimicrobials, and consumer preferences regarding medication-free poultry production have pushed the poultry industry toward reducing the use of AGP in poultry feed [3–5]. Other methods used to control coccidiosis, such as vaccination with live Eimeria vaccines, may also have an indirect effect on the incidence of NE [3]. The reduction in the use of AGP in poultry diets due to antibiotic bans in Europe has resulted in an increase of antibiotics used in treatment of flocks
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induce apoptosis. Instead, the Ca influx induced a cascade of events consistent with programmed necrosis, as it was associated with calpain activation and release of cathepsins from lysosomes. In addition, they also observed deregulation of mitochondrial activity leading to an increase in reactive oxygen species and dramatically decreasing ATP levels [53]. These findings strongly suggest that cellular death from poreforming toxins is related to a programmed cellular necrosis. Strong evidence exists that netB is an essential virulence mediator for the development of NE. Initial screening of poultry NE isolates found that the majority of the isolates (77%) were netB-positive [49, 52]. In addition, C. perfringens isolates that were unable to cause NE were analyzed for the presence of the netB gene and most of these isolates (91.2%) were found to be netB-negative [49]. Keyburn et al. [52] reported that C. perfringens netB knockout isolates were not able to cause NE, whereas the original netB-positive isolates were able to cause disease. Therefore, although a clear association exists between netB and NE development, as not all C. perfringens isolates from NE outbreaks were netB-positive, other virulence factors may be present that play an important role in the onset and development of NE. The presence of the netB gene in isolates from healthy birds also suggests that the presence of netB is not sufficient to cause disease, which reveals the importance of predisposing factors for the development of NE.
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as virginiamycin-treated birds. Lactobacillus spp. are also promising candidates to be used as probiotics in commercial poultry. In a study conducted to evaluate the effectiveness of a direct-fed microbial containing Lactobacillus acidophilus and Lactobacillus casei, cecal counts of C. perfringens were significantly lower when poults were fed the probiotic in comparison to control birds [59]. In another study using Lactobacillus fermentum as a probiotic, NE lesion severity of birds fed probiotic was significantly reduced when compared with challenged birds not fed probiotics [60]. In the same study, authors reported that probiotic supplementation significantly downregulated the levels of Tolllike receptor 2 and IFN-γ and upregulated the expression of IL-10 [60]. Another class of feed additives being researched with the objective of improving intestinal health is phytogenics and plant-derived compounds. Phytogenics include a broad range of plant materials, and essential oils represent a particular subcategory of phytogenics [57]. The addition of phytogenic products to bird feeds has shown antimicrobial action, which is attributed to the ability of phytogenics to disintegrate bacterial cell membrane and penetrate bacterial cells. These antimicrobial properties are associated with the lipophilic character of phytogenics [57]. Birds fed diets containing phytogenic blends presented significantly reduced severity of NE-associated lesions and mortality due to NE when compared with birds that were fed diets without the phytogenic blends [56]. In the same study, when birds were fed a combination of the phytogenic blends with a multispecies probiotic, the combination of feed additives significantly reduced NE lesion severity when compared with birds fed control diets. However, no improvements in lesion severity were observed when birds were fed the combination of the feed additives in comparison to feeding birds probiotics or phytogenic blend separately [56]. Research targeting the use of alternative feed additives to help treat and prevent NE have reported inconsistent results. Geier et al. [61] compared the efficiency of different feed additives in preventing and treating NE. The feed additives tested were a blend of organic acids (formic, acetic, propionic, sorbic, caprylic and capric acids), direct-fed microbials (Lactobacil-
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with NE [54]. Necrotic enteritis has been treated by administering lincomycin, bacitracin, oxytetracycline, penicillin, and tylosin in water [4, 7]. Bacitracin, lincomycin, virginiamycin, penicillin, avoparcin, and nitrovin can also be used in the feed to treat NE [4, 7]. Necrotic enteritis vaccine studies show inconsistent results for effective methods of NE prevention. Most vaccination efforts have been directed to producing toxoid vaccines by using culture supernatant, of which α-toxin is the major component [6, 45, 49]. Recent findings suggesting that netB, and not α-toxin, is the main virulence factor in the pathogenesis of NE were thought to explain the inconsistency of these results. Strong evidence suggesting that netB could be used as a toxoid and offer better protection than α-toxin has been noted [6, 49, 55]. Lanckriet et al. [55] reported that vaccination of birds with supernatant from 2 different netB-positive strains of C. perfringens significantly protected birds against NE. However, in the same study, no protection against NE was observed when vaccinating birds with the toxoid of the other 3 strains of C. perfringens that were also netB positive. These results indicate that immunity to NE induced after vaccination with supernatant of C. perfringens is not entirely determined by netB or α-toxin expression, but probably involves other antigens that have not been identified. Feed additives, such as probiotics, are becoming popular prevention tools for NE. Probiotics are composed of beneficial microorganims that are administered to birds with the intent of modulating the intestinal microbiota. The full mechanism by which probiotics help balance the intestinal microbiota is not fully understood and varies depending on the probiotic used. However, it is suggested that beneficial bacteria in probiotic products modulate the intestinal microbiota by competing for nutrients and attachment sites with pathogenic bacteria, producing natural antibiotics, and stimulating the immune system [56, 57]. Bacillus licheniformis has been researched as a direct-fed microbial with the potential to prevent enteric disease and reduce the severity of ongoing enteritis. Knap et al. [58] reported that the addition of 8 × 107 cfu/g of feed of B. licheniformis was able to reduce NE mortality and lesion score to the same level
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CONCLUSIONS AND APPLICATIONS
1. Necrotic enteritis is a complex disease that is very important to the commercial poultry industry because of the economic cost associated with infected flocks. The complexity of NE pathogenesis makes modeling, treating, and preventing this disease a real challenge. The challenge certainly increases with the reduction of the use of AGP in poultry diets. 2. Efforts in identifying the synergic effects of different virulence factors, such as netB and α-toxin, and their mechanisms of action will definitely aid in solving the NE puzzle. Additionally, it is important to research the synergic effects among aforementioned virulence factors with predisposing factors, such as nutrient levels and dietary ingredients, so nutritionists can effectively formulate diets for its effects in gastrointestinal health. 3. Identifying mechanisms that cause C. perfringens to overgrow in the intestine
of poultry and cause NE is essential to control this disease. 4. In conclusion, NE remains a challenge to the poultry industry, and this challenge is becoming greater each day, with more strict regulations and consumers pushing for a product produced with lower levels of AGP. Although advances in NE research have contributed to identifying predisposing factors and preventative resources for NE, research projects focused on identifying the complete pathogenesis of NE and the mode of action of alternative feed ingredients are necessary to effectively prevent and treat NE without the aid of antibiotics.
REFERENCES AND NOTES 1. Van der Sluis, W. 2000. Clostridial enteritis is an often underestimated problem. World’s Poult. Sci. J. 16:42– 43. 2. McReynolds, J. L., J. A. Byrd, R. C. Anderson, R. W. Moore, T. S. Edrington, K. J. Genovese, T. L. Poole, L. F. Kubena, and D. J. Nisbet. 2004. Evaluation of immunosupressants and dietary mechanisms in an experimental disease model for necrotic enteritis. Poult. Sci. 83:1948–1952. 3. Van Immerseel, F., J. D. Buck, F. Pasmans, G. Huyghebaert, F. Haesebrouck, and R. Ducatelle. 2004. Clostridium perfringens in poultry: An emerging threat for animal and public health. Avian Pathol. 33:537–549. 4. Opengart, K. 2008. Necrotic enteritis. Pages 872–877 in Diseases of Poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. McDougald, L. K. Nolan, and D. E. Swayne, ed. Wiley-Blackwell Publishing, Ames, IA. 5. Williams, R. B. 2005. Intercurrent coccidiosis and necrotic enteritis of chickens: Rational, integrated disease management by maintenance of gut integrity. Avian Pathol. 34:159–180. 6. Lee, K. W., H. S. Lillehoj, M. S. Park, S. I. Jang, G. D. Ritter, Y. H. Hong, W. Jeong, H. Y. Jeoung, D. J. An, and E. P. Lillehoj. 2012. Clostridium perfringens alpha-toxin and netB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens. Avian Dis. 56:230–233. 7. Cooper, K. K., and J. G. Songer. 2009. Necrotic enteritis in chickens: A paradigm of enteric infection by Clostridium perfringens type A. Anaerobe 15:55–60. 8. Smith, L. D. S. 1992. The genus Clostridium—Medical. Pages 1867–1878 in The Prokaryotes: A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, vol 2, 2nd ed. A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K. Schleifer, ed. Springer-Verlag Inc., New York, NY. 9. McClane, B. 2001. Clostridium perfringens. Pages 351–372 in Food Microbiology: Fundamentals and Frontiers, 2nd ed. M. P. Doyle, L. R. Beuchat, and T. J. Montville, ed. ASM Press, Washington, DC.
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lus johnsonii), and antimicrobials (zinc bacitracin and monensin). Those authors did not report any differences in mortality due to NE or NE lesion score averages when comparing birds supplemented with the probiotic, the organic acid blend, and the challenged control. However, birds fed the antimicrobial presented significantly lower mortality and NE lesion score averages when compared with all other treatments [61]. The inconsistency of results involving the use of alternative feed additives to AGP may be related to differences in the inclusion levels and mode of action of different feed additives. Additionally, the variability of the results might also be related to the virulence of C. perfringens strains used in different studies associated with a disease that is difficult to model and reproduce experimentally. Results could also be influenced by the multitude of other contributing factors for NE occurrence. Benefits from probiotic and phytogenic blends are usually observed when NE episodes are mild. However, when severe episodes of NE are reproduced experimentally, supplementation benefits are usually masked by the severity of the disease.
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tinal barrier function in a chick model of necrotic enteritis. Antimicrob. Agents Chemother. 47:3311–3317. 27. Collier, C. T., C. L. Hofacre, A. M. Payne, D. B. Anderson, P. Kaiser, R. I. Mackie, and H. R. Gaskins. 2008. Coccidia induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth. Vet. Immunol. Immunopathol. 122:104–115. 28. Drew, M. D., N. A. Syed, B. G. Goldade, B. Laarveld, and A. G. van Kessel. 2004. Effects of dietary protein source and levels on intestinal populations of Clostridium perfringens in broiler chickens. Poult. Sci. 83:414–420. 29. Guillouard, I., P. M. Alzari, B. Saliou, and S. Cole. 1997. The carboxy-terminal C2-like domain of the alphatoxin from Clostridium perfringens mediates calcium-dependent membrane recognition. Mol. Microbiol. 26:867– 876. 30. Titball, R. W., C. Naylor, and A. Basak. 1999. The Clostridium perfringens alpha-toxin. Anaerobe 5:51–64. 31. Selle, P. H., A. J. Cowieson, and V. Ravindran. 2009. Consequences of calcium interaction with phytate and phytase for poultry and pigs. Livest. Sci. 124:126–141. 32. Walk, C. L., M. R. Bedford, and A. P. McElroy. 2012b. Influence of limestone and phytase on broiler performance, gastrointestinal pH, and apparent ileal nutrient digestibility. Poult. Sci. 91:1371–1378. 33. Wages, D. P., and K. Opengart. 2003. Necrotic enteritis. Pages 781–785 in Diseases of Poultry, 11th ed. Y.M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald, and D. E. Swayne, ed. Iowa State Press, Ames. 34. Paiva, D. M., C. Walk, and A. McElroy. 2013. Influence of dietary calcium level, calcium source, and phytase on bird performance and mineral digestibility during a natural necrotic enteritis episode. Poult. Sci. 92:3125–3133. 35. Tsiouris, V., I. Georgopoulu, C. Batzious, N. Papaioannou, P. Fortomaris, and R. Ducatelle. 2009. Effects of heat stress on the pathogenesis of necrotic enteritis in broiler chickens. Pages 149–153 in Proc. 2nd Mediterr. Summit World’s Poult. Sci. Assoc., Antalaya, Turkey. 36. Kmet, V., M. Stachova, R. Nemcova, Z. Jonecova, and A. Laukova. 1993. The interaction of intestinal microflora with avian enteric pathogens. Acta Veterinaria 62:S87–S89. 37. Lillejoh, H. S., and J. Trout. 1996. Avian gut-associated lymphoid tissues and intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev. 9:349–360. 38. Songer, J. G. 1996. Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev. 9:216–234. 39. Petit, L., M. Gibert, and M. R. Popoff. 1999. Clostridium perfringens: Toxinotype and genotype. Trends Microbiol. 7:104–110. 40. Moreau, H., G. Pieroni, C. Jolivet-Reynaud, J. E. Alouf, and R. Verger. 1988. A new kinetic approach for studying phospholipase C (Clostridium perfringens alphatoxin) activity on phospholipid monolayers. Biochemistry 27:2319–2323. 41. Sakurai, J., M. Nagahama, and M. Oda. 2004. Clostridium perfringens alpha-toxin: Characterization and mode of action. J. Biochem. 136:569–574. 42. Al-Sheikhly, F., and R. B. Truscott. 1977. The pathology of necrotic enteritis of chickens following infusion of crude toxins of Clostridium perfringens into the duodenum. Avian Dis. 21:241–255. 43. Hofshagen, M., and H. Stenwig. 1992. Toxin production by Clostridium perfringens isolated from broiler chick-
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10. Wrigley, D. M. 2001. Clostridium perfringens. Pages 139–168 in Foodborne Disease Handbook, Volume 1: Bacterial Pathogens, 2nd ed. H. Y. Hui, M. D. Pierson, and J. R. Gorham, ed. Marcel Dekker Inc., New York, NY. 11. Setlow, P., and E. A. Johnson. 2001. Spores and their significance. Pages 33–70 in Food Microbiology: Fundamentals and Frontiers, 2nd ed. M. P. Doyle, L. R. Beuchat, and T. J. Montville, ed. ASM Press, Washington, DC. 12. Si, W., J. Gong, Y. Han, H. Yu, J. Brennan, H. Zhou, and S. Chen. 2007. Quantification of cell proliferation and alpha-toxin gene expression of Clostridium perfringens in the development of necrotic enteritis in broiler chickens. Appl. Environ. Microbiol. 73:7110–7113. 13. Craven, S. E., N. A. Cox, N. J. Stern, and J. M. Mauldin. 2001. Prevalence of Clostridium perfringens in commercial broiler hatcheries. Avian Dis. 45:1050–1053. 14. Timbermont, L., F. Haesebrouck, R. Ducatelle, and F. V. Immerseel. 2011. Necrotic enteritis in broilers: An updated review on the pathogenesis. Avian Pathol. 40:341–347. 15. 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 Poult. Sci. J. 62:221–247. 16. Lee, K. W., H. S. Lillehoj, W. Jeong, H. Y. Jeoung, and D. J. An. 2011. Avian necrotic enteritis: Experimental models, host immunity, pathogenesis, risk factors, and vaccine development. Poult. Sci. 90:1381–1390. 17. Branton, S. L., F. N. Reece, and W. M. Hagler Jr. 1987. Influence on a wheat diet on mortality of broiler chickens associated with necrotic enteritis. Poult. Sci. 66:1326–1330. 18. Riddell, C., and X. M. Kong. 1992. The influence of diet on necrotic enteritis in broiler chickens. Avian Dis. 36:499–503. 19. Antoniou, T., R. R. Marquardt, and P. E. Cansfield. 1981. Isolation, partial characterization, and antinutritional activity of a factor in rye grain. J. Agric. Food Chem. 29:1240–1247. 20. Hesselman, K., and P. Aman. 1986. The effect of bglucanase on the utilization of starch and nitrogen by broiler chickens fed on barley of low or high viscosity. Anim. Feed Sci. Technol. 15:83–93. 21. Ward, A. T., and R. R. Marquardt. 1987. Antinutritional activity of a water-soluble pentosan-rich fraction form rye grain. Poult. Sci. 66:1665–1674. 22. Fengler, A., and R. R. Marquardt. 1988. Water-soluble pentosans from rye: II. Effects on rate of dialysis and on the retention of nutrients by the chick. Cereal Chem. 65:298–302. 23. Langhout, D. J., J. B. Schutte, P. V. 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. Br. Poult. Sci. 40:340–347. 24. 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 challenge. Poult. Sci. 88:132–140. 25. Craven, S. E., N. A. Cox, N. J. Stern, and J. M. Mauldin. 2001. Prevalence of Clostridium perfringens in commercial broiler hatcheries. Avian Dis. 45:1050–1053. 26. Collier, C. T., J. D. Klis, B. Deplancke, D. B. Anderson, and H. R. Gaskins. 2003. Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intes-
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ens and capercaillies with and without necrotizing enteritis. Avian Dis. 36:837–843. 44. Lovland, A., M. Kaldhusdal, K. Redhead, E. Skjerve, and A. Lillehaug. 2004. Maternal vaccination against subclinical necrotic enteritis in broilers. Avian Pathol. 33:83– 92. 45. Van Immerseel, F., J. I. Rood, R. Moore, and R. Titball. 2009. Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens. Trends Microbiol. 17:32–36. 46. Keyburn, A. L., S. Sheedy, M. Ford, M. Williamson, M. Awad, J. Rood, and R. J. Moore. 2006. Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens. Infect. Immun. 74:6496– 6500. 47. Olkowski, A. A., C. Wojnarowicz, M. Chirino-Tejo, and M. D. Drew. 2006. Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis. Res. Vet. Sci. 81:99–108. 48. Bryant, A. E., C. R. Bayer, M. J. Adalpe, R. J. Wallace, R. W. Titball, and D. L. Stevens. 2006. Clostridium perfringens phospholipase C-induced platelet/leukocyte interactions impede neutrophil diapedesis. J. Med. Microbiol. 55:495–504. 49. Keyburn, A. L., T. L. Bannam, R. J. Moore, and J. I. Rood. 2010. NetB, a pore-forming toxin from necrotic enteritis strains of C. perfringens. Toxins 2:1913–1927. 50. Savva, C. G., S. P. F. da Costa, M. Bokori-Brown, C. E. Naylor, A. R. Cole, D. S. Moss, R. W. Titball, and A. Basak. 2013. Molecular architecture and functional analysis of netB, a pore forming toxin from Clostridium perfringens. J. Biol. Chem. 288:3512–3522. 51. Yan, X. X., C. J. Porter, S. P. Hardy, D. Steer, A. Smith, N. Quinsey, V. Hughes, J. K. Cheung, A. Keyburn, M. Kaldhusdal, R. Moore, T. Bannam, J. C. Whisstock, and J. I. Rood. 2013. Structural and functional analysis of the pore-forming toxin netB from Clostridium perfringens. MBio 4:e00019–e00013. 52. Keyburn, A.L., J.D. Boyce, P. Vaz, T. Bannam, M. Ford, D. Parker, A. Rubbo, J. Rood, and R.J. Moore. 2008.
JAPR: Review Article
Necrotic enteritis: Applications for the poultry industry Diego Paiva and Audrey McElroy1
Primary Audience: Researchers, Veterinarians, Nutritionists, Live Production Managers SUMMARY Considering market demands concerning the decreased use of growth promoters and anticoccidial drugs in feed formulations, the poultry industry has been trying to reduce or eliminate the inclusion of subtherapeutic doses of antimicrobials into feed. Formulating diets not only to meet birds’ nutrient requirements for growth but also for gastrointestinal health parameters is increasingly important. Maintenance and enhancement of intestinal integrity is essential for bird performance when antimicrobials are not included in feed, as commercial poultry face numerous enteric pathogen challenges. Necrotic enteritis has reemerged as an important disease of poultry in recent years. The reduction in the use of antimicrobials in poultry feeds has been attributed as one of the main contributing factors for the increasing incidence of necrotic enteritis (NE) in commercial poultry. Mortality due to NE is extremely high (1% daily mortality), which results in great economic losses. Economic losses due to NE are not only associated with high mortality, but also associated with decreases in bird performance and FE, particularly in subclinical cases of NE. Birds that survive NE outbreaks usually have a reduced ability to digest and absorb nutrients due to extensive damage to the mucosal lining, which ultimately results in reduced profitability. Key words: necrotic enteritis, Clostridium perfringens, netB, alpha-toxin, chicken, enteritis 2014 J. Appl. Poult. Res. 23:557–566 http://dx.doi.org/10.3382/japr.2013-00925
INTRODUCTION Necrotic enteritis (NE) is an enterotoxemia of poultry with an important economic effect on poultry production. It has been estimated that NE costs the poultry industry $2 billion globally as a result of reduction in bird performance and disease treatment [1, 2]. The reduction in bird performance is not only associated with impaired growth rate and feed conversion during production, but also with increased condemna-
1
Corresponding author: [email protected]
tion rates in broilers due to hepatitis at the processing plant [3, 4]. In the past, the use of antimicrobial growth promoters (AGP) in commercial poultry feed helped control NE in poultry flocks [5, 6]. However, NE has reemerged as a significant problem in poultry production, likely as a result of national and international policies that ban or limit the use of AGP in poultry feeds [4, 5, 7]. In addition, consumers’ preferences have had a large effect on bird production, and the push for poultry
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Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061
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558 production with less medication in the diet has also had a significant effect on the increased incidence of NE in the past few years [7]. The objective of the current review is to discuss recent findings in the pathogenesis of NE and alternative prevention and treatment options.
ETIOLOGY AND EPIDEMIOLOGY
PREDISPOSING FACTORS Clostridium perfringens is a naturally occurring bacterium in the intestines of warmblooded animals, and its presence alone is not a determining factor for disease development. Therefore, predisposing factors that could lead to an overgrowth of C. perfringens are crucial to NE onset and development. Several predisposing factors for NE have been identified, including dietary factors, immune status and stress, intestinal physiopathology, and coccidiosis. Dietary Factors The gastrointestinal tract ecosystem is very susceptible to diet composition. Different inclusion levels of specific nutrients can have a significant effect on the microbial population in the intestine and ultimately affect bird performance and susceptibility to diseases. Several different dietary components may favor C. perfringens growth and consequently NE onset and development. The type of cereal grain used in poultry diets has been identified as a dietary component affecting the onset of NE. Research has shown that the use of diets formulated with cereals (barley, rye, oats, and wheat) containing high levels of indigestible, water-soluble, nonstarch polysaccharides (NSP) predisposes birds to NE [17, 18]. Whereas the mechanism by which NSP predisposes birds to NE is not clear, the effects that NSP have on bird performance, nutrient digestibility, and digesta viscosity have been extensively reported. Complex carbohydrates are known to have a negative effect on bird performance [19, 20]. The reduction in performance can be associated with a decrease in nutrient digestibility of several nutrients, such as amino acids, fat, and cholesterol, as well as decreased DM retention [20–22]. These nutrients then
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Clostridium perfringens (types A and C) is the etiological agent associated with NE [4]. Clostridium perfringens is a gram-positive, rodshaped, anaerobic encapsulated bacterium that causes a broad spectrum of human and veterinary diseases [8–10]. Clostridium perfringens differs from many other clostridia in being nonmotile, reducing nitrate, and carrying out stormy fermentation of lactose in milk [10, 11]. Clostridium perfringens also ferments glucose, fructose, galactose, inositol, maltose, mannose, starch, and sucrose. The fermentation products include acetic and butyric acids with or without butanol [8]. Clostridium perfringens will grow over a wide pH range, varying from 5.5 to 8.5, whereas optimum growth of this bacterium occurs at pH 6 to 7 [9, 11]. The virulence of C. perfringens largely results from its prolific toxinproducing ability. The classification scheme for C. perfringens assigns isolates to 1 of the 5 types (A–E) depending upon their ability to express 1 or all 4 “typing” toxins (α, β, ε, and ι) [8–10]. Clostridium perfringens has a wide distribution, being isolated from the intestine of warmblooded animals and a variety of environments, such as soil, water, and feed [3, 8]. Colonization in the intestines of poultry may happen as early as the day of hatch in the hatcheries. It has been reported that 105 cfu/g of C. perfringens in ileal digesta is the threshold for the development of NE in chickens [12]. Craven et al. [13] were able to isolate C. perfringens from eggshell fragments, chicken fluff, and paper pads in commercial broiler hatcheries. Given that C. perfringens is a naturally occurring bacterium in the intestinal environment of poultry, disease development is dependent on other predisposing factors that will be discussed further in the current review. Although chickens from 2 wk to 6 mo of age are susceptible to NE, incidence varies greatly according to type of bird and rearing conditions.
Most NE outbreaks in broiler flocks raised on litter are reported between the second and fifth week of age [4, 5, 14]. In contrast, NE outbreaks in commercial layers raised in floor pen settings have been reported between 3 to 6 mo of age [4]. Mortality rates associated to NE in broilers are often between 2 and 10%; however, rates as high as 50% have been reported [15, 16].
Paiva and McElroy: NECROTIC ENTERITIS REVIEW
Mineral levels in the diet have also been identified as dietary factors that could predispose birds to NE. Zinc was the first mineral associated with NE through its involvement α-toxin activity. The α-toxin produced by C. perfringens depends on zinc to hydrolyze membrane lipids [15, 29, 30]. This mechanism is discussed in detail in the C. perfringens toxins section of the current review. Dietary levels of Ca have also been identified as a relevant nutrient for the pathogenesis of NE, but the mechanism by which Ca favors C. perfringens is unknown. It has been suggested that excess Ca may increase intestinal pH [31, 32] and favor C. perfringens growth in a more neutral environment [5, 33]. Paiva et al. [34] reported that mortality due to NE was significantly higher when feeding broilers 0.9% Ca in the diet when compared with birds fed 0.6% Ca in the diet. However, differences in gastrointestinal pH were not observed in their study when feeding 2 different levels of Ca in the diet during naturally occurring NE, suggesting that Ca was required for α-toxin and netB activity through mechanisms that will be further discussed in the present review. Immune Status and Stress Immunosuppression predisposes animals to NE, because the factors that usually lead to immunosupression likely alter the intestinal environment and the intestinal microbial population [14, 15]. Pathogens that lead to immunosuppression (Eimeria spp., infectious bursal disease virus, chick anemia virus, and Marek’s disease virus) have been reported to predispose chickens to NE [2, 16]. In an NE disease model study, McReynolds et al. [2] reported that birds infected with infectious bursal disease virus often have secondary infections with C. perfringens. Stress likely has the same immunosuppressive effects of the aforementioned pathogens. Environmental (heat or cold stress) and managerial (feed changes, litter conditions, stocking density, vaccination programs) stressors have been shown to cause immunosupression, predisposing birds to disease [2, 15, 35]. Intestinal Physiopathology When intestinal conditions are not favorable, even highly virulent C. perfringens strains fail
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become available to microbial utilization in the lower small intestine and could potentially change the type and balance of intestinal microbiota [22, 23]. Jia et al. [24] reported that digesta was more viscous in birds consuming diets containing high levels of NSP. The increase in digesta viscosity due to NSP has been associated with an increase in mucous secretion [23] and an increase in water-binding capacity by complex carbohydrates [19, 22]. High digesta viscosity impairs nutrient digestion by interfering with the interaction between digestive enzymes and their substrates and impeding nutrient uptake in the gastrointestinal tract [19, 20, 22]. As a result, an influx of undigested nutrients enters the distal segments of the small intestine increasing microbial growth. The increased mucous secretion results in a nutrient rich environment favoring C. perfringens over other bacterial species [23], as this bacterium is known to have mucolytic activity and a short generation time [25–27]. Dietary protein levels and protein source are also factors that can predispose birds to the development of NE. Protein-rich diets result in a high concentration of protein in the gastrointestinal tract that will serve as substrate for microbial growth [14]. In addition, in the small intestine, proteins are degraded to nitrogenous compounds (ammonia and amines) that not only increase intestinal pH (high logarithmic acid dissociation constant of nitrogenous compounds), but also favor the proliferation of pathogenic bacteria, including C. perfringens [8, 15]. Evidence suggests that protein source may be more important than the protein levels in the diet for NE development. High inclusion levels of animal protein sources (fishmeal, meat and bone meal) are usually associated with an increase in the incidence of NE in broilers [28]. Timbermont et al. [14] hypothesized that animal protein sources would have higher levels of indigestible protein that would reach the ceca and serve as substrate for C. perfringens. Another possibility is that the amino acid balance (especially methionine and glycine) in these feed ingredients would ultimately favor C. perfringens growth in the ceca, leading to NE [5, 28]. However, it is still unclear why high inclusion levels of animal protein in the diet of broilers results in an increase in the ocurrence of NE.
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Coccidiosis Coccidiosis is among the most studied predisposing factors of NE. Coccidiosis in chickens is usually caused by the association of 2 or more species of Eimeria: Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox, and Eimeria tenella. Coccidia oocysts are ubiquitous in areas where poultry are raised, allowing this parasite to cycle and remain in the environment. The Eimeria spp. life cycle is complex and involves stages inside and outside of the host. The consequence of the Eimeria cycle is extensive damage to the intestinal mucosa. The intestinal lumen then becomes rich with plasma proteins, which are themselves rich in amino acids, growth factors, and vitamins that serve as substrate for clostridial growth [3, 5, 14]. In addition, digestibility of nutrients is reduced due to extensive gastrointestinal damage, which may substantially increase nutrient availability for C. perfringens [5, 37]. Furthermore, coccidiosis induces
a local T cell-mediated inflammatory response that increases mucin (and mucous) production [27, 37], which ultimately favors C. perfringens growth due to its mucolytic ability [26, 27]. Clostridium perfringens Toxins Clostridium perfringens is an extracellular pathogen, thus its virulence is mostly associated with its ability to produce toxins. Clostridium perfringens can produce up to 17 toxic or potentially toxic exoproteins [38]. The species is divided into types (A, B, C, D, and E) according to the production of 4 major toxins: α, β, ε, and ι. Type A comprises the strains that produce α-toxin, type B as strains that produce α, β, and ε toxins, type C as strains that produce α and β toxins, type D as strains that produce α and ε toxins, and type E as strains that produce α and ι toxins [38, 39]. As only C. perfringens types A and C are associated with NE, only α and netB toxins will be discussed in the present review. Alpha-Toxin Alpha-toxin is a zinc-dependent phospholipase sphingomyelinase C and is encoded by the phospholipase C gene [7, 29, 30]. Alpha-toxin is organized in 2 distinct domains: the N-terminal region (247 residues), which carries the active site required for phospholipid hydrolysis, and the C-terminal region (123 residues), which carries the lipid-binding site [29, 39]. Alpha-toxin requires zinc for substrate hydrolysis [15, 29, 30], and Ca ions are essential for the binding of α-toxin to lipid films [30, 39, 40]. High Ca concentrations are required for optimal enzymatic activity with physiological substrates [29, 41]. Alpha-toxin has been shown to be cytotoxic, leading to cell lysis of erythrocytes, phagocytes, fibroblasts, platelets, leukocytes, endothelial cells, and myocytes [29, 38]. Alpha-toxin hydrolyses phospholipids and promotes cellular membrane disorganization. In vivo, the cellular substrates for α-toxin are phosphatidylcholine and sphingonyelin, which are both components of the cellular membrane of epithelial cells in the gastrointestinal system [15, 30]. The mechanism of membrane recognition is a complex event involving Ca-mediated phospholipid recognition, where Ca ions are
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to produce disease. However, some intestinal physiopathological circumstances exist that favor the development of NE, such as intestinal stasis, changes in gastrointestinal pH, and damage to the intestinal mucosa [5, 7, 16]. When intestinal motility is reduced (i.e., increased digesta viscosity) feed passage is delayed. This increases nutrient availability to the microbial population in the gastrointestinal tract, allowing C. perfringens to outgrow other species [5]. Associated with an increase in nutrient availability, an increase in transit time also means a reduction in microbial flushing from the gastrointestinal tract, which increases the opportunity of pathogens to proliferate, colonize, and cause disease. Intestinal acidity can also have an effect on NE onset. Higher intestinal pH can predispose birds to develop NE, as C. perfringens growth is inhibited in more acidic intestines [36]. Damage of the intestinal mucosa is another condition that predisposes broilers to develop NE. This predisposition is likely a combination of effects, such as changes in intestinal pH, excess nutrients in the gastrointestinal tract, leakage of plasma proteins and growth factors to the intestinal environment, and loss of intestinal integrity.
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not the main virulence factor of NE. One of the hallmarks of NE in broilers is granulocyte migration from the tissue to the intestinal lumen [47]. This inflammatory reaction is very different from the leukostasis and lack of inflammatory response induced by α-toxin in gas gangrene [48], indicating that NE lesions are probably mediated by toxins other than α-toxin. NetB Toxin NetB, another toxin associated with NE occurrence in broilers, is a pore-forming toxin, with similarity to C. perfringens β-toxin (38% identity), C. perfringens gamma-toxin (40% identity), Staphylococcus aureus α-hemolysin (30% identity), and Staph. aureus gamma-toxin (23% identity) [49, 50]. NetB forms discrete pores of at least 1.6 nm in diameter in the cellular membrane, causing an influx of ions (Ca, Na, Cl, and so on) that eventually lead to osmotic cell lysis [49, 50]. The entrances of the pores formed by netB are negatively charged, which explains why netB pores are cation-selective [50, 51]. NetB is secreted as a water-soluble monomer that is thought to oligomerize on the target cell surface before forming pores [52]. Although Savva et al. [50] reported that netB is able to oligomerize and form pores in the absence of cholesterol, the conversion to oligomer and pore formation is significantly increased in the presence of cholesterol. It was initially suggested that birds were more susceptible to NE than mammals due to differences in cholesterol concentration in the cell membrane of birds. Yan et al. [51] reported differences in susceptibility of avian and mammalian red blood cells to netB mediated lysis. However, as avian and mammalian red blood cells have similar proportions of cell membrane cholesterol, these differences seem to be unrelated to cholesterol levels. The exact role of membrane cholesterol in netB function is still unclear. In addition to promoting cell lysis through an osmotic imbalance, the Ca influx caused by netB may also lead to a special type of programmed cell death. Kennedy et al. [53] reported that the pore-forming α-toxin of Clostridium septicum forms Ca-permeable pores, which increase intracellular Ca. What was surprising about their findings was the fact that the Ca influx did not
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partially coordinated by acidic amino acid side chains of α-toxin and partially by phosphate groups or membrane phospholipids [30]. Hydrolysis of cell membrane phospholipids results in the formation of diacylglycerol, following an activation of protein kinase C and consequent activation of arachidonic acid cascade [15, 39, 41]. The outcome of the activation of the arachidonic acid cascade is the synthesis of inflammatory mediators (leukotrienes, tromboxane, platelet-agglutinating factor, and prostacyclin), which cause blood vessel contraction, platelet aggregation, and myocardial dysfunction, leading to acute death [3, 39, 41]. Alpha-toxin has been indicated as the main virulence mediator for NE in poultry. Al-Sheikhly and Truscott [42] were able to successfully reproduce NE in birds infused intraduodenally with bacteria-free crude toxin. Another indication of α-toxin participation in NE pathogenesis is that C. perfringens isolates from broilers with NE produce significantly more α-toxin than isolates from broilers without NE [43]. Studies of broiler immune response to NE also suggest that α-toxin is an important virulence factor in the pathogenesis of NE. Broilers with NE have significantly lower antibody levels against α-toxin than healthy controls [6, 44]. Higher levels of toxin-reactive antibodies in healthy chickens, when compared with chickens with NE symptoms, indicate that these antibodies may protect birds from developing NE. Despite these reports, the role of α-toxin as the main virulence factor for producing NE in broilers is a very controversial topic. Interpretation of early results can be disputed because most of the studies that reported α-toxin as the main virulence factor in the pathogenesis of NE used crude supernatant instead of purified α-toxin [42, 43]. Conclusions of these studies were based on α-toxin being the most dominant protein present in crude supernatants and did not consider the presence of other toxins in crude supernatants that might be collaborating or even responsible for the development of NE [45]. The most convincing evidence that α-toxin is not the main virulence mediator in the development of NE comes from Keyburn et al. [46], where an α-toxin-negative mutant of a C. perfringens strain from an NE outbreak was still able to produce NE in broilers. Other factors of NE pathogenesis also indicate that α-toxin is
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CLINICAL SIGNS AND PATHOLOGY Necrotic enteritis clinical signs are common to enteritis in general: depression, anorexia, diarrhea, dehydration, and ruffled feathers [3, 4, 7]. The classic acute form of NE is characterized by a sudden increase in flock mortality without any warning clinical signs [3, 14]. The subclinical form of NE is usually mild with no clinical signs or peak in mortality; most of the time, only an overall reduction in bird performance is observed [3, 43]. Performance losses are associated with chronic intestinal mucosal damage resulting in poor nutrient digestion and absorption, reduced BW gain, and increased FCR [3, 14]. In cases of subclinical NE, an increase in
liver condemnations at the processing plant is often observed due to cholangiohepatitis [3, 14]. Therefore, the subclinical form of NE is harder to diagnose and birds are not treated, resulting in greater economic losses [14]. Macroscopic lesions are usually restricted to the jejunum and ileum, but may extend to the duodenum and ceca [4, 7, 14]. The small intestine is usually distended and filled with gas; intestinal walls are thin and friable [4, 14]. Necrosis is visible in the intestinal mucosa, along with presence of a green to yellow diphtheritic membrane that is adherent to the mucosa [4]. Histopathology reveals a severe inflammatory response to C. perfringens. Inflammatory infiltrate is characterized by the presence of heterophils, lymphocytes, macrophages, and plasma cells [14, 47]. Diffuse and severe coagulative necrosis of the mucosa is also observed [14, 47]. Masses of tissue fragments, necrotic cells, cell debris, and bacterial colonies comprise the diphteric membrane characteristic of NE [14, 47]. Blood vessel congestion can be observed in the lamina propria and submucosa.
PREVENTION AND TREATMENT Necrotic enteritis prevention is usually associated with management practices that minimize the effects of the predisposing factors that contribute to disease development. Reducing the inclusion of dietary ingredients that may lead to NE, such as fish meal, oats, barley, and rye, has been a noteworthy solution in decreasing NE incidence [7]. The use of AGP in feed has also played an important role in the control of NE. The introduction of AGP in the diet assists with coccidiosis management and modifies the intestinal microbial populations, which both result in a reduction in the incidence of NE [3, 7]. However, government bans, pathogen resistance to antimicrobials, and consumer preferences regarding medication-free poultry production have pushed the poultry industry toward reducing the use of AGP in poultry feed [3–5]. Other methods used to control coccidiosis, such as vaccination with live Eimeria vaccines, may also have an indirect effect on the incidence of NE [3]. The reduction in the use of AGP in poultry diets due to antibiotic bans in Europe has resulted in an increase of antibiotics used in treatment of flocks
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induce apoptosis. Instead, the Ca influx induced a cascade of events consistent with programmed necrosis, as it was associated with calpain activation and release of cathepsins from lysosomes. In addition, they also observed deregulation of mitochondrial activity leading to an increase in reactive oxygen species and dramatically decreasing ATP levels [53]. These findings strongly suggest that cellular death from poreforming toxins is related to a programmed cellular necrosis. Strong evidence exists that netB is an essential virulence mediator for the development of NE. Initial screening of poultry NE isolates found that the majority of the isolates (77%) were netB-positive [49, 52]. In addition, C. perfringens isolates that were unable to cause NE were analyzed for the presence of the netB gene and most of these isolates (91.2%) were found to be netB-negative [49]. Keyburn et al. [52] reported that C. perfringens netB knockout isolates were not able to cause NE, whereas the original netB-positive isolates were able to cause disease. Therefore, although a clear association exists between netB and NE development, as not all C. perfringens isolates from NE outbreaks were netB-positive, other virulence factors may be present that play an important role in the onset and development of NE. The presence of the netB gene in isolates from healthy birds also suggests that the presence of netB is not sufficient to cause disease, which reveals the importance of predisposing factors for the development of NE.
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as virginiamycin-treated birds. Lactobacillus spp. are also promising candidates to be used as probiotics in commercial poultry. In a study conducted to evaluate the effectiveness of a direct-fed microbial containing Lactobacillus acidophilus and Lactobacillus casei, cecal counts of C. perfringens were significantly lower when poults were fed the probiotic in comparison to control birds [59]. In another study using Lactobacillus fermentum as a probiotic, NE lesion severity of birds fed probiotic was significantly reduced when compared with challenged birds not fed probiotics [60]. In the same study, authors reported that probiotic supplementation significantly downregulated the levels of Tolllike receptor 2 and IFN-γ and upregulated the expression of IL-10 [60]. Another class of feed additives being researched with the objective of improving intestinal health is phytogenics and plant-derived compounds. Phytogenics include a broad range of plant materials, and essential oils represent a particular subcategory of phytogenics [57]. The addition of phytogenic products to bird feeds has shown antimicrobial action, which is attributed to the ability of phytogenics to disintegrate bacterial cell membrane and penetrate bacterial cells. These antimicrobial properties are associated with the lipophilic character of phytogenics [57]. Birds fed diets containing phytogenic blends presented significantly reduced severity of NE-associated lesions and mortality due to NE when compared with birds that were fed diets without the phytogenic blends [56]. In the same study, when birds were fed a combination of the phytogenic blends with a multispecies probiotic, the combination of feed additives significantly reduced NE lesion severity when compared with birds fed control diets. However, no improvements in lesion severity were observed when birds were fed the combination of the feed additives in comparison to feeding birds probiotics or phytogenic blend separately [56]. Research targeting the use of alternative feed additives to help treat and prevent NE have reported inconsistent results. Geier et al. [61] compared the efficiency of different feed additives in preventing and treating NE. The feed additives tested were a blend of organic acids (formic, acetic, propionic, sorbic, caprylic and capric acids), direct-fed microbials (Lactobacil-
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with NE [54]. Necrotic enteritis has been treated by administering lincomycin, bacitracin, oxytetracycline, penicillin, and tylosin in water [4, 7]. Bacitracin, lincomycin, virginiamycin, penicillin, avoparcin, and nitrovin can also be used in the feed to treat NE [4, 7]. Necrotic enteritis vaccine studies show inconsistent results for effective methods of NE prevention. Most vaccination efforts have been directed to producing toxoid vaccines by using culture supernatant, of which α-toxin is the major component [6, 45, 49]. Recent findings suggesting that netB, and not α-toxin, is the main virulence factor in the pathogenesis of NE were thought to explain the inconsistency of these results. Strong evidence suggesting that netB could be used as a toxoid and offer better protection than α-toxin has been noted [6, 49, 55]. Lanckriet et al. [55] reported that vaccination of birds with supernatant from 2 different netB-positive strains of C. perfringens significantly protected birds against NE. However, in the same study, no protection against NE was observed when vaccinating birds with the toxoid of the other 3 strains of C. perfringens that were also netB positive. These results indicate that immunity to NE induced after vaccination with supernatant of C. perfringens is not entirely determined by netB or α-toxin expression, but probably involves other antigens that have not been identified. Feed additives, such as probiotics, are becoming popular prevention tools for NE. Probiotics are composed of beneficial microorganims that are administered to birds with the intent of modulating the intestinal microbiota. The full mechanism by which probiotics help balance the intestinal microbiota is not fully understood and varies depending on the probiotic used. However, it is suggested that beneficial bacteria in probiotic products modulate the intestinal microbiota by competing for nutrients and attachment sites with pathogenic bacteria, producing natural antibiotics, and stimulating the immune system [56, 57]. Bacillus licheniformis has been researched as a direct-fed microbial with the potential to prevent enteric disease and reduce the severity of ongoing enteritis. Knap et al. [58] reported that the addition of 8 × 107 cfu/g of feed of B. licheniformis was able to reduce NE mortality and lesion score to the same level
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CONCLUSIONS AND APPLICATIONS
1. Necrotic enteritis is a complex disease that is very important to the commercial poultry industry because of the economic cost associated with infected flocks. The complexity of NE pathogenesis makes modeling, treating, and preventing this disease a real challenge. The challenge certainly increases with the reduction of the use of AGP in poultry diets. 2. Efforts in identifying the synergic effects of different virulence factors, such as netB and α-toxin, and their mechanisms of action will definitely aid in solving the NE puzzle. Additionally, it is important to research the synergic effects among aforementioned virulence factors with predisposing factors, such as nutrient levels and dietary ingredients, so nutritionists can effectively formulate diets for its effects in gastrointestinal health. 3. Identifying mechanisms that cause C. perfringens to overgrow in the intestine
of poultry and cause NE is essential to control this disease. 4. In conclusion, NE remains a challenge to the poultry industry, and this challenge is becoming greater each day, with more strict regulations and consumers pushing for a product produced with lower levels of AGP. Although advances in NE research have contributed to identifying predisposing factors and preventative resources for NE, research projects focused on identifying the complete pathogenesis of NE and the mode of action of alternative feed ingredients are necessary to effectively prevent and treat NE without the aid of antibiotics.
REFERENCES AND NOTES 1. Van der Sluis, W. 2000. Clostridial enteritis is an often underestimated problem. World’s Poult. Sci. J. 16:42– 43. 2. McReynolds, J. L., J. A. Byrd, R. C. Anderson, R. W. Moore, T. S. Edrington, K. J. Genovese, T. L. Poole, L. F. Kubena, and D. J. Nisbet. 2004. Evaluation of immunosupressants and dietary mechanisms in an experimental disease model for necrotic enteritis. Poult. Sci. 83:1948–1952. 3. Van Immerseel, F., J. D. Buck, F. Pasmans, G. Huyghebaert, F. Haesebrouck, and R. Ducatelle. 2004. Clostridium perfringens in poultry: An emerging threat for animal and public health. Avian Pathol. 33:537–549. 4. Opengart, K. 2008. Necrotic enteritis. Pages 872–877 in Diseases of Poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. McDougald, L. K. Nolan, and D. E. Swayne, ed. Wiley-Blackwell Publishing, Ames, IA. 5. Williams, R. B. 2005. Intercurrent coccidiosis and necrotic enteritis of chickens: Rational, integrated disease management by maintenance of gut integrity. Avian Pathol. 34:159–180. 6. Lee, K. W., H. S. Lillehoj, M. S. Park, S. I. Jang, G. D. Ritter, Y. H. Hong, W. Jeong, H. Y. Jeoung, D. J. An, and E. P. Lillehoj. 2012. Clostridium perfringens alpha-toxin and netB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens. Avian Dis. 56:230–233. 7. Cooper, K. K., and J. G. Songer. 2009. Necrotic enteritis in chickens: A paradigm of enteric infection by Clostridium perfringens type A. Anaerobe 15:55–60. 8. Smith, L. D. S. 1992. The genus Clostridium—Medical. Pages 1867–1878 in The Prokaryotes: A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, vol 2, 2nd ed. A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K. Schleifer, ed. Springer-Verlag Inc., New York, NY. 9. McClane, B. 2001. Clostridium perfringens. Pages 351–372 in Food Microbiology: Fundamentals and Frontiers, 2nd ed. M. P. Doyle, L. R. Beuchat, and T. J. Montville, ed. ASM Press, Washington, DC.
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lus johnsonii), and antimicrobials (zinc bacitracin and monensin). Those authors did not report any differences in mortality due to NE or NE lesion score averages when comparing birds supplemented with the probiotic, the organic acid blend, and the challenged control. However, birds fed the antimicrobial presented significantly lower mortality and NE lesion score averages when compared with all other treatments [61]. The inconsistency of results involving the use of alternative feed additives to AGP may be related to differences in the inclusion levels and mode of action of different feed additives. Additionally, the variability of the results might also be related to the virulence of C. perfringens strains used in different studies associated with a disease that is difficult to model and reproduce experimentally. Results could also be influenced by the multitude of other contributing factors for NE occurrence. Benefits from probiotic and phytogenic blends are usually observed when NE episodes are mild. However, when severe episodes of NE are reproduced experimentally, supplementation benefits are usually masked by the severity of the disease.
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