NetB Toxin and Immunization Against Necrotic Enteritis in Poultry: A Comprehensive Review

Document Type : Review


1 Department of anaerobic bacterial vaccine Production and Research, Clostridia Research Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

2 Department of Pathobiology, School of Veterinary Medicine, ShahreKord University, ShahreKord, Iran

3 3Department of Biotechnology, Institute of Applied Sciences and Humanities, GLA University, Mathura, India


Necrotic enteritis, caused by avian-specific NetB toxin-producing strains of Clostridium perfringens type A, has gained worldwide concerns due to increased production losses and mortality in broilers, increased veterinarian costs, and the chance of getting contamination in products for human consumption. Prevention strategies include supplementing diet or drinking water with conventional therapeutic antibiotic growth promoters and
anti-coccidial drugs.. There are various strategies to prevent the disease, including antibiotic growth promoters. Antibiotic growth promoters are antimicrobial agent that used for control of diseases such as necrotic enteritis. Other factor for prevention, anti-coccidial drugs that are taken orally. However, vaccination against toxin-producing C. perfringens type A via nano, toxoid, genetically modified, or other clostridial vaccines is a effective preventive against necrotic enteritis. This comprehensive review describes the recent advances in the development of NetB vaccines, other strategies to enhance immunization, their delivery systems in poultry against necrotic enteritis, and their pathogenesis. This review also exlains future immunization strategies like breeder hen vaccination, in ovo vaccination, and live (attenuated) vectors to be used in feed additives and other predisposing factors applicable in the field. All the vaccines discussed in the manuscript have shown their effectiveness against necrotic enteritis in poultry.


Main Subjects

Adams CA. 2004. Nutricines in poultry production: focus on bioactive feed ingredients. Paper presented at the Nutrition Abstracts and Reviews. Series B, Livestock Feeds and Feeding. CAB International. Wallingford. UK. Pages, 1N-12N. DOI: 10.1079/cabireviews20043109361
Akerele G, Ramadan N, Renu S, Renukaradhya GJ, Shanmugasundaram R & Selvaraj RK. 2020. In vitro characterization and immunogenicity of chitosan nanoparticles loaded with native and inactivated extracellular proteins from a field strain of Clostridium perfringens associated with necrotic enteritis. Veterinary Immunology and Immunopathology, 224: 110059. DOI: 10.1016/j.vetimm.2020.110059
Asif M, Jenkins KA, Hilton LS, Kimpton WG, Bean AG & Lowenthal JW. 2004. Cytokines as adjuvants for avian vaccines. Immunology and Cell Biology, 82(6): 638-643. DOI: 10.1111/j.1440-1711.2004.01295.x
Atterbury RJ, Morris V, Harrison D, Tucker V, Allen V, Davies R & Carrique-Mas J. 2010. Effect of delivery method on the efficacy of Salmonella vaccination in chickens. Veterinary Record, 167(5): 161-164. DOI: 10.1136/vr.b4884
Cooper KK, Songer JG & Uzal FA. 2013. Diagnosing clostridial enteric disease in poultry. Journal of Veterinary Diagnostic Investigation, 25(3): 314-327. DOI: 10.1177/1040638713483468
da Costa SPF, Mot D, Bokori-Brown M, Savva CG, Basak AK, Van Immerseel F & Titball RW. 2013. Protection against avian necrotic enteritis after immunisation with NetB genetic or formaldehyde toxoids. Vaccine, 31(37): 4003-4008. DOI: 10.1016/j.vaccine.2013.05.063
da Costa SPF, Mot D, Geeraerts S, Bokori-Brown M, Van Immerseel F & Titball RW. 2016. Variable protection against experimental broiler necrotic enteritis after immunization with the C-terminal fragment of Clostridium perfringens alpha-toxin and a non-toxic NetB variant. Avian Pathology, 45(3): 381-388. DOI: 10.1080/03079457.2015.1129663
da Costa SPF, Savva CG, Bokori-Brown M, Naylor CE, Moss DS, Basak AK & Titball RW. 2014. Identification of a key residue for oligomerisation and pore-formation of Clostridium perfringens NetB. Toxins, 6(3): 1049-1061. DOI: 10.3390/toxins6031049
Duff AF, Vuong CN, Searer KL, Briggs WN, Wilson KM, Hargis BM, Berghman LR & Bielke LR. 2019. Preliminary studies on development of a novel subunit vaccine targeting Clostridium perfringens mucolytic enzymes for the control of necrotic enteritis in broilers. Poultry Science, 98(12): 6319-6325. DOI: 10.3382/ps/pez448
Elwinger K a, Schneitz C, Berndtson E, Fossum O, Teglöf B & Engstöm B. 1992. Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks. Acta Veterinaria Scandinavica, 33(4): 369-378. DOI: 10.1186/BF03547304
Gangaiah D, Ryan V, Van Hoesel D, Mane SP, Mckinley ET, Lakshmanan N, Reddy ND, Dolk E & Kumar A. 2022. Recombinant Limosilactobacillus (Lactobacillus) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis. Microbiology Open, 11(2): e1270. DOI: 10.1002/mbo3.1270
Gardiner DF, Rosenberg T, Zaharatos J, Franco D & Ho DD. 2009. A DNA vaccine targeting the receptor-binding domain of Clostridium difficile toxin A. Vaccine, 27(27): 3598-3604. DOI: 10.1016/j.vaccine.2009.03.058
Hegazy WAH & Hensel M. 2012. Salmonella enterica as a vaccine carrier. Future Microbiology, 7(1): 111-127. DOI: 10.2217/fmb.11.144
Islam M, Rashid S, Juli M, Hoque M & Akter M. 2009. Necrotic enteritis in chickens: Pathological, bacteriological and therapeutical investigation. International Journal of Sustainable Crop Production, 4(3): 1-7.
Jang SI, Lillehoj HS, Lee SH, Lee KW, Lillehoj EP, Hong YH, An DJ, Jeong W, Chun JE, Bertrand F & Dupuis L. 2012. Vaccination with Clostridium perfringens recombinant proteins in combination with Montanide™ ISA 71 VG adjuvant increases protection against experimental necrotic enteritis in commercial broiler chickens. Vaccine, 30(36): 5401-5406. DOI: 10.1016/j.vaccine.2012.06.007
Jiang Y, Mo H, Willingham C, Wang S, Park JY, Kong W, Roland KL & Curtiss III R. 2015. Protection against necrotic enteritis in broiler chickens by regulated delayed lysis Salmonella vaccines. Avian Diseases, 59(4): 475-485. DOI: 10.1637/11094-041715-Reg
Jin K, Wang S, Zhang C, Xiao Y, Lu S & Huang Z. 2013. Protective antibody responses against Clostridium difficile elicited by a DNA vaccine expressing the enzymatic domain of toxin B. Human Vaccines & Immunotherapeutics, 9(1): 63-73. DOI: 10.4161/hv.22434
 Kaldhusdal M, Schneitz C, Hofshagen M & Skjerve E. 2001. Reduced incidence of Clostridium perfringens-associated lesions and improved performance in broiler chickens treated with normal intestinal bacteria from adult fowl. Avian Diseases, 149-156. DOI: 10.2307/1593022
Katalani C, Nematzadeh G, Ahmadian G, Amani J, Kiani G & Ehsani P. 2019. Design and expression optimization of a chimeric derivative of NetB, Alpha-toxin and metallopeptidase proteins as a subunit vaccine against Clostridium perfringens. Vaccine Research, 6: 5-12.
Keyburn AL, Boyce JD, Vaz P, Bannam TL, Ford ME, Parker D, Di Rubbo A, Rood JI & Moore RJ. 2008. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathogens, 4(2): e26. DOI: 10.1371/journal.ppat.0040026
Keyburn AL, Portela RW, Ford ME, Bannam TL, Yan XX, Rood JI & Moore RJ. 2013a. Maternal immunization with vaccines containing recombinant NetB toxin partially protects progeny chickens from necrotic enteritis. Veterinary Research, 44(1): 108. DOI: 10.1186/1297-9716-44-108
Keyburn AL, Portela RW, Sproat K, Ford ME, Bannam TL, Yan X, Rood JI & Moore RJ. 2013b. Vaccination with recombinant NetB toxin partially protects broiler chickens from necrotic enteritis. Veterinary Research, 44(1): 54. DOI: 10.1186/1297-9716-44-54
Keyburn AL, Yan XX, Bannam TL, Van Immerseel F, Rood JI & Moore RJ. 2010. Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin. Veterinary Research, 41(2): 1-8. DOI: 10.1051/vetres/2009069
Kong W, Clark-Curtiss J & Curtiss III R. 2013. Utilizing Salmonella for antigen delivery: the aims and benefits of bacterial delivered vaccination. Expert Review of Vaccines, 12(4): 345-347. DOI: 10.1586/erv.13.7
Kulkarni R, Parreira V, Jiang YF & Prescott J. 2010. A live oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing Clostridium perfringens antigens confers protection against necrotic enteritis in broiler chickens. Clinical and Vaccine Immunology, 17(2): 205-214. DOI: 10.1128/CVI.00406-09
Kulkarni R, Parreira V, Sharif S & Prescott J. 2007. Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis. Clinical and Vaccine Immunology, 14(9): 1070-1077. DOI: 10.1128/CVI.00162-07
Kulkarni R, Parreira V, Sharif S & Prescott J. 2008. Oral immunization of broiler chickens against necrotic enteritis with an attenuated Salmonella vaccine vector expressing Clostridium perfringens antigens. Vaccine, 26(33): 4194-4203. DOI: 10.1016/j.vaccine.2008.05.079
Lanckriet A, Timbermont L, Eeckhaut V, Haesebrouck F, Ducatelle R & Van Immerseel F. 2010. Variable protection after vaccination of broiler chickens against necrotic enteritis using supernatants of different Clostridium perfringens strains. Vaccine, 28(36): 5920-5923. DOI: 10.1016/j.vaccine.2010.06.035
Lee KW, Lillehoj HS, Park MS, Jang SI, Ritter GD, Hong YH, Jeong W, Jeoung HY, An DJ & Lillehoj EP. 2012. Clostridium perfringens α-toxin and NetB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens. Avian Diseases, 56(1): 230-233. DOI: 10.1637/9847-070711-ResNote.1
Lee K, Lillehoj HS, Li G, Park MS, Jang SI, Jeong W, Jeoung HY, An DJ & Lillehoj EP. 2011. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate: ferredoxin oxidoreductase (PFO) of C. perfringens. Research in Veterinary Science, 91(3): e80-e86. DOI: 10.1016/j.rvsc.2011.01.017
Lepp D, Roxas B, Parreira VR, Marri PR, Rosey EL, Gong J, Songer JG, Vedantam G & Prescott JF. 2010. Identification of novel pathogenicity loci in Clostridium perfringens strains that cause avian necrotic enteritis. PloS One, 5(5): e10795. DOI: 10.1371/journal.pone.0010795
Li J, Adams V, Bannam TL, Miyamoto K, Garcia JP, Uzal FA, Rood JI & McClane BA. 2013. Toxin plasmids of Clostridium perfringens. Microbiology and Molecular Biology Reviews, 77(2): 208-233. DOI: 10.1128/MMBR.00062-12
Li N, Yu YZ, Yu WY & Sun ZW. 2011. Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM-CSF gene adjuvant. Immunopharmacology and Immunotoxicology, 33(1): 211-219. DOI: 10.3109/08923971003782327
Lillehoj HS, Jang SI, Panebra A, Lillehoj EP, Dupuis L, Arous JB, Lee SK & Oh ST. 2017. In ovo vaccination using Eimeria profilin and Clostridium perfringens NetB proteins in Montanide IMS adjuvant increases protective immunity against experimentally-induced necrotic enteritis. Asian-Australasian Journal of Animal Sciences, 30(10): 1478. DOI:  10.5713/ajas.17.0053
Lovland A, Kaldhusdal M, Redhead K, Skjerve E & Lillehaug A. 2004. Maternal vaccination against subclinical necrotic enteritis in broilers. Avian Pathology, 33(1): 81-90. DOI: 10.1080/0379450310001636255
Mishra N & Smyth JA. 2017. Oral vaccination of broiler chickens against necrotic enteritis using a non-virulent NetB positive strain of Clostridium perfringens type A. Vaccine, 35(49): 6858-6865. DOI: 10.1016/j.vaccine.2017.10.030
Muir W, Bryden W & Husband A. 2000. Immunity, vaccination and the avian intestinal tract. Developmental & Comparative Immunology, 24(2-3): 325-342. DOI:  10.1016/s0145-305x(99)00081-6
Ohtani K & Shimizu T. 2016. Regulation of toxin production in Clostridium perfringens. Toxins, 8(7): 207. DOI: 10.3390/toxins8070207
Paap P & Additives O. 2015. Dietary treatments for major poultry diseases.
Ramadan NM, Akerele G, Renu S, Renukaradhya G & Selvaraj R. 2020. Oral immunization of broilers with chitosan nano-encapsulated extracellular and cell wall proteins of necrotic enteritis-induced Clostridium perfringens. BioRxiv. DOI: 10.1101/2020.10.23.351817
Rappuoli R, Black S & Lambert PH. 2011. Vaccine discovery and translation of new vaccine technology. The Lancet, 378(9788): 360-368. DOI: 10.1016/S0140-6736(11)60440-6
 Ricks CA, Avakian A, Bryan T, Gildersleeve R, Haddad E, Ilich R, King S, Murray L, Phelps P, Poston R & Whitfill C. 1999. In ovo vaccination technology. Advances in Veterinary Medicine, 41: 495.
Riddell C & Kong X-M. 1992. The influence of diet on necrotic enteritis in broiler chickens. Avian Diseases, 36: 499-503. DOI: 10.2307/1591740
Robinson K, Chamberlain L, Lopez M, Rush C, Marcotte H, Le Page R & Wells J. 2004. Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains expressing tetanus toxin fragment C. Infection and Immunity, 72(5): 2753-2761. DOI: 10.1128/IAI.72.5.2753-2761.2004
Rood JI, Keyburn AL & Moore RJ. 2016. NetB and necrotic enteritis: the hole movable story. Avian Pathology, 45(3): 295-301. DOI: 10.1080/03079457.2016.1158781
Rostami A, Goshadrou F, Langroudi RP, Bathaie SZ, Riazi A, Amani J & Ahmadian G. 2016. Design and expression of a chimeric vaccine candidate for avian necrotic enteritis. Protein Engineering, Design and Selection, 30(1): 39-45. DOI: 10.1093/protein/gzw060
Saikh KU, Sesno J, Brandler P & Ulrich RG. 1998. Are DNA-based vaccines useful for protection against secreted bacterial toxins? Tetanus toxin test case. Vaccine, 16(9-10): 1029-1038. DOI: 10.1016/S0264-410X(97)00280-6
Sasaki J, Goryo M, Honda J, Okoshi N, Okada K & Furukawa H. 2000. Cholangiohepatitis in broiler chickens in Japan: histopathological, immunohistochemical and microbiological studies of spontaneous disease. Acta Veterinaria Hungarica, 48(1): 59-67. DOI: 10.1556/AVet.48.2000.1.7
Savva CG, da Costa SP, Bokori-Brown M, Naylor CE, Cole AR, Moss DS, Titball RW & Basak AK. 2013. Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens. Journal of Biological Chemistry, 288(5): 3512-3522. DOI: 10.1074/jbc.M112.430223
Schijns VE, van de Zande S, Lupiani B & Reddy SM. 2014. Practical aspects of poultry vaccination. Avian Immunology, 288(5): 345-362. DOI: 10.1016/B978-0-12-396965-1.00020-0
Sharma J. 1999. Introduction to poultry vaccines and immunity. Advances in Veterinary Medicine, 41: 481-494. DOI: 10.1016/s0065-3519(99)80036-6.
Song L, Hobaugh M R, Shustak C, Cheley S, Bayley H & Gouaux J E. 1996. Structure of staphylococcal α-hemolysin, a heptameric transmembrane pore. Science, 274(5294): 1859-1865. DOI: 10.1126/science.274.5294.1859
Timbermont L, Haesebrouck F, Ducatelle R & Van Immerseel F. 2011. Necrotic enteritis in broilers: an updated review on the pathogenesis. Avian Pathology, 40(4): 341-347. DOI: 10.1080/03079457.2011.590967
Wang S, Kong Q & Curtiss III R. 2013. New technologies in developing recombinant attenuated Salmonella vaccine vectors. Microbial Pathogenesis, 58: 17-28. DOI: 10.1016/j.micpath.2012.10.006
Wilde S, Jiang Y, Tafoya AM, Horsman J, Yousif M, Vazquez LA & Roland KL. 2019. Salmonella-vectored vaccine delivering three Clostridium perfringens antigens protects poultry against necrotic enteritis. PloS One, 14(2): e0197721. DOI: 10.1371/journal.pone.0197721
Williams R. 2005. Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathology, 34(3): 159-180. DOI: 10.1080/03079450500112195
Yan XX, Porter CJ, Hardy SP, Steer D, Smith AI, Quinsey NS, Hughes V, Cheung JK, Keyburn AL, Kaldhusdal M & Moore RJ. 2013. Structural and functional analysis of the pore-forming toxin NetB from Clostridium perfringens. MBio, 4(1): e00019-00013. DOI: 10.1128/mBio.00019-13
Zaragoza NE, Orellana CA, Moonen GA, Moutafis G & Marcellin E. 2019. Vaccine Production to Protect Animals Against Pathogenic Clostridia. Toxins, 11(9): 525. DOI: 10.3390/toxins11090525
Zekarias B, Mo H & Curtiss, R. 2008. Recombinant attenuated Salmonella enterica serovar typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens. Clinical and Vaccine Immunology, 15(5): 805-816. DOI:  10.1128/CVI.00457-07