Clostridium perfringens Necrotic Enteritis B-like Toxin: Comparison
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Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type A/G that impacts the global poultry industry by compromising the performance, health, and welfare of chickens. Coccidiosis is a major contributing factor to NE. Although NE pathogenesis was believed to be facilitated by α-toxin, a chromosome-encoded phospholipase C enzyme, studies have indicated that NE B-like (NetB) toxin, a plasmid-encoded pore-forming heptameric protein, is the primary virulence factor. Since the discovery of NetB toxin, the occurrence of NetB+ C. perfringens strains has been increasingly reported in NE-afflicted poultry flocks globally. It is generally accepted that NetB toxin is the primary virulent factor in NE pathogenesis although scientific evidence is emerging that suggests other toxins contribute to NE. Because of the complex nature of the host-pathogen interaction in NE pathogenesis, the interaction of NetB with other potential virulent factors of C. perfringens needs better characterization.

  • lostridium perfringens
  • necrotic enteritis
  • NetB toxin
  • pathogenesis
  • NE detection
  • broiler chickens

1. Introduction

The world population is rapidly expanding and is expected to reach over nine billion people by the year 2050 [1]. Most of this population growth is expected to occur in Africa and Asia where the poultry industry will be required to sustainably meet the increasing demand for safer poultry meat and eggs. Thus, our role as animal scientists in combating world hunger is critical and there is a timely need to develop strategic priorities for sustainable food production systems. The global poultry industry is a dynamic industry that has successfully met an ever-increasing demand on global poultry meat and egg consumption. There are, however, ongoing challenges [2] to solve for sustainable poultry and eggs production. Those challenges include legislative bans on antibiotic use in animal feed, global climate change, and economically important disease outbreaks affecting the food industry, all of which could negatively affect optimum poultry performance. For example, it was estimated that a higher ambient temperature lowered the productivity and welfare of chickens resulting in an economic loss of USD128 to 165 million for the US poultry industry [3].
Over the last 50 years, sub-therapeutic doses of in-feed antibiotics have been a reliable tool to increase the welfare and productivity of chickens by controlling pathogenic bacteria and preventing dysbacteriosis. However, increasing concerns regarding the occurrence of antibiotic-resistant bacteria and drug residues in poultry meats halted the use of antibiotics in poultry feed globally. Finally, enteric disorders caused by Eimeria spp. or Clostridium perfringens, both of which are ubiquitous in the environment and gastrointestinal tract of animals, compromise the health, performance, and welfare of chickens. Coccidiosis caused by Eimeria spp. results in an economic loss of about 13 billion US dollars per year due to mortality and the use of in-feed anticoccidials [4]. Controlling enteric disorders is expected to be more challenging in the post-antibiotic era, and there is a timely need to develop novel solutions to promote the health and performance of chickens. Topics on gut health and alternatives to antibiotic use in chickens have been discussed elsewhere [2,5,6,7,8,9,10][2][5][6][7][8][9][10].

2. Clostridium perfringens Toxinotypes

C. perfringens is anaerobic, non-motile, Gram-positive spore-forming bacteria found ubiquitously, including as a normal microbiota in animals, and produces at least 20 virulent toxins and enzymes [20,21][11][12]. C. perfringens is a fast-growing bacterium with an 8–12 min generation time when cultured at 43 °C in optimal media that extends to 12–17 min at 37 °C. Once dominantly colonized under optimal environments (e.g., predisposing factors), the fast-growing C. perfringens can cause a rapid onset of NE [22][13]. Six virulent factors (e.g., toxins/enzymes) have been used to designate the strains from A to G depending on their production capability for α-toxin, β-toxin, ε-toxin, ι-toxin, enterotoxin, and NE toxin beta-like (NetB) toxin [21][12] (Table 1), and their functions and production have been reported [20,22,23][11][13][14]. Initially, C. perfringens had five toxinotypes based on the chromosomal presence of α-toxin, β-toxin, ε-toxin, or ι-toxin. However, the discovery of new toxin genes (e.g., enterotoxin and netB) and toxin-specific C. perfringens-mediated diseases has led to the classification of seven C. perfringens toxinotypes (Table 1). In the new toxinotyping scheme, C. perfringens type F produces α-toxin and enterotoxin but not β-toxin, ε-toxin, ι-toxin, or NetB toxin, and is an important enteric pathogen that causes food poisoning in humans [21,24][12][15]. This toxinotyping scheme emphasizes the role of enterotoxin production in C. perfringens-mediated food poisoning cases.
Table 1.
Revised toxin-based classification of
Clostridium perfringens
.
Type Toxin A Produced (Structural Gene)
α-Toxin (cpa) β-Toxin (cpb) ε-Toxin (etx) ι-Toxin (iap) CPE (cpe) NetB (netB)
A + − − − − −
B + + + − − −
C + + − − ± −
D + − + − ± −
E + − − + ± −
F + − − − + −
G + − − − − +
A Symbol (+) indicates the presence of the toxin and symbol (−) indicates the absence of the toxin.
All toxinotypes (i.e., type A to G) of C. perfringens can extracellularly secrete high levels of α-toxin, which is encoded by the chromosomal plc gene (Table 1). α-Toxin is a zinc-metalloenzyme with a phospholipase C (plc) domain that is responsible for phospholipase, sphingomyelinase, and hemolytic activities [22][13]. Thus, α-toxin has long been considered a major virulence factor in NE necrotic lesions. However, the recently discovered NetB toxin by C. perfringens is now proposed to be the primary toxin responsible for NE pathogenesis in broilers [25][16]. Accordingly, C. perfringens type G is responsible for NE in chickens as it has genes encoding the two typing toxins α-toxin and NetB [21][12]. On the other hand, C. perfringens type A can only produce α-toxin, but not β-toxin, ε-toxin, ι-toxin, enterotoxin, or NetB toxin. One exception to the seven toxinotype scheme is one reported case that showed the netB gene in C. perfringens type C, which was isolated from a subclinical NE case [26][17]. Nonetheless, the netB gene is only detected in C. perfringens type G strains.

3. Incidence of NetB-Positive C. perfringens Isolated from Broilers

Since the discovery of the NetB toxin as an essential virulence factor in NE, the prevalence of netB-positive C. perfringens isolates in NE disease outbreaks has been reported (Table 42). In general, culture methods utilizing media and agar were used to isolate C. perfringens from chicken digesta or litter samples prior to the netB genotyping with polymerase chain reaction (PCR) or quantitative PCR. C. perfringens can be directly isolated by plating gut or environmental samples on selective agar (e.g., TSC agar) or by a liquid culture (e.g., FTG, CMM, or BHI) followed by plating on solid agar (e.g., TSC, blood, or BHI). The function of selective media for C. perfringens has been documented elsewhere [71][18] although their contributions to growth and toxin production have not studied.
Table 42. Incidence of necrotic enteritis (NE) B-like toxin gene in Clostridium perfringens G isolates from retail chicken meats or chickens afflicted with or without clinical or subclinical NE.
Country Study Year A Detection Method NE Chicken, n/Total Healthy Chicken, n/Total Ref.
NetB Positive % NetB Positive %
Australia 2010 PCR 14/18 77.8 - - [25][16]
Australia/Canada/Belgium/Denmark 2010 PCR 31/44 70.5 2/55 3.6 [85][19]
Brazil 2012 PCR 0/22 0.0 - - [86][20]
Canada 2005–2007 PCR 39/41 95.1 7/20 35.0 [87][21]
Canada 2011–2012 PCR 9/45 20.0 12/18 66.7 [62][22]
Canada 2011–2012 PCR 41/41 100.0 26/30 86.7 [62][22]
Canada 2010 PCR 6/6 100.0 4/5 80.0 [66][23]
Canada B 2010 PCR - - 39/183 21.3 [88][24]
Denmark 1997–2002 PCR 13/25 52.0 14/23 60.9 [60][25]
Denmark/

Finland		 1997–2001 PCR 12/22 54.5 0/8 0.0 [89][26]
Iran 2016 PCR 8/45 17.8 - - [90][27]
Italy C 2015–2017 qPCR - - 31/151 20.5 [65][28]
Italy 2009 PCR 16/30 53.3 4/22 18.2 [91][29]
Korea 2010–2012 PCR 8/17 47.1 2/50 4.0 [61][30]
Korea B 2018 PCR - - 4/9 44.4 [92][31]
Netherlands 2012 PCR 43/45 95.6 - - [26][17]
Sweden 2004 PCR 31/34 91.2 - D 25.0 [93][32]
Sweden 2004 PCR 16/23 69.6 - - [93][32]
Sweden 2004 PCR 0/11 0.0 - - [93][32]
USA 2004–2009 PCR 17/20 85.0 10/54 18.5 [67][33]
USA 2009 PCR 7/12 58.3 7/80 8.8 [59][34]
USA 2018 qPCR 11/15 73.3 9/15 60.0 [63][35]
USA 2016 PCR 119/145 82.1 59/85 69.4 [64][36]
USA 2003–2004 PCR 19/19 100.0 - - [47][37]
A = year of publication was provided unless the studied year was not indicated. B = retail chicken meats. C = flock surveillance with no health status. D = not specified.
 
Several conclusions can be drawn from studies that describe C. perfringens isolation from NE-afflicted or healthy chickens (Table 42): (1) Field NE occurs globally, (2) netB-positive C. perfringens are isolated from both NE-afflicted and healthy chickens, (3) netB-positive C. perfringens is more abundant in NE-afflicted vs. healthy chickens, (4) netB-positive C. perfringens may not be isolated from confirmed NE clinical cases, (5) netB-negative C. perfringens can be isolated from NE-afflicted chickens, and (6) netB-positive C. perfringens can be detected in retail broiler meats. Thus, these findings indicate that NE is a complex disease caused by multiple contributing and predisposing factors (e.g., C. perfringens toxins, Eimeria, stress, nutrition, or environment) although the NetB toxin is considered to be the primary virulent factor.

4. Concluding Remarks

NE is not only an emerging threat to the global broiler industry but also compromises the welfare and health of commercial poultry in the era of antibiotic-independent poultry production [12,97][38][39]. This threat will be enhanced under the “No Antibiotics Ever (NAE)” and “Antibiotic-free” (ABF) broiler production systems. Thus, understanding NE pathogenesis and developing sensitive detection assay are important to reduce economic losses due to NE and to develop early management strategies against NE. With the development of molecular and proteomic technologies, the NetB toxin has been regarded as the primary virulence factor in NE pathogenesis. However, new scientific evidence is emerging to implicate other C. perfringens toxins and enzymes that can potentiate NE pathogenesis with or without NetB. Although the presence of netB in C. perfringens isolates can be typed with PCR and the NetB protein can be detected using Western blots, there is a need for a more defined large-scale immunoassay that can detect toxins associated with NE pathogenesis in the field samples from NE-afflicted farms. Recent availability of NetB-specific mouse monoclonal antibodies and a high throughput ELISA detection system will now allow sensitive NetB detection in samples from poultry farms. These NetB-specific monoclonal antibody-based detection assays will be a valuable tool in addressing fundamental questions of host-pathogen immunobiology in NE and as a screening tool for validating the efficacy of novel alternative-to-antibiotics feed additives or vaccines to mitigate the negative effects of NE.

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