Skip to main content
SpringerLink
Log in

Bacteriophage mediated control of necrotic enteritis caused by C. perfringens in broiler chickens.

  • Original Article
  • Published:
Veterinary Research Communications Aims and scope Submit manuscript

Abstract

In Egypt, little attention has been paid to the isolation and application of C. perfringens phages for treating necrotic enteritis at the farm level. This study aims to evaluate the efficiency of the podovirus C. perfringens phage in treating necrotic enteritis in broiler chickens. Accordingly, C. perfringens phage was isolated from cecal samples of apparently healthy chickens and characterized by transmission electron microscopy, thermal stability test, and pH stability test. Commercial 14-day-old Arbor Acres broiler chickens were allocated to three groups: group Ӏ received BHI broth and assigned as a negative control, group П served as a positive control group that was challenged with C. perfringens via oral gavage for four successive days, and group Ш was administrated six phage doses on several occasions after oral gavage challenge with C. perfringens. Daily clinical symptoms and mortality were recorded. At three-time intervals, necrotic enteritis lesions were scored. Cecal samples were examined for re-isolation and counting of C. perfringens. The isolated C. perfringens phage was a podovirus with an icosahedral head diameter of 78.7 nm and a short non-contractile tail length of 22.2 nm. It remained stable for 60 min at 30 °C and 50 °C at pH values of 2, 4, 8, and 10. The phage-treated group (Ш) showed mild gross lesions with a lower mortality rate and reduced colony-forming units than the positive control group (П). The findings revealed that the isolated C. perfringens phage effectively treated experimental necrotic enteritis in broiler chickens.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Ackermann HW (2009) Basic phage electron microscopy, in: Clokie, M.R.J., Kropinski, Andrew (Ed.), bacteriophages: methods and protocols, volume 1: isolation, characterization, and interactions. Human press, springer, New York, NY pp. 113-126

  • Arbor Acres Plus Broiler Nutrition Specifications. (2014). Retrieved from http://www.aviagen.com

  • Apajalahti JH, Kettunen A, Bedford MR, Holben WE (2001) Percent G+ C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens. Appl Environ Microbiol 67:5656–5667

    Article  CAS  Google Scholar 

  • Bae D, Lee JW, Chae JP, Kim JW, Eun JS, Lee KW, Seo KH (2021) Characterization of a novel bacteriophage φCJ22 and its prophylactic and inhibitory effects on necrotic enteritis and Clostridium perfringens in broilers. Poult Sci 100:302–313

    Article  CAS  Google Scholar 

  • Carrillo CML, Connerton PL, Pearson T, Connerton IF (2007) Free-range layer chickens as a source of Campylobacter bacteriophage. Antonie Van Leeuwenhoek 92:275–284

    Article  Google Scholar 

  • CLSI (2017) Performance standards for antimicrobial susceptibility testing: twenty-seventh informational supplement. CLSI document M100-S27. Clinical and laboratory standards institute Wayne, PA

  • Cooper KK, Songer JG (2009) Necrotic enteritis in chickens: a paradigm of enteric infection by Clostridium perfringens type a. Anaerobe 15:55–60

    Article  Google Scholar 

  • Cooper KK, Songer JG (2010) Virulence of Clostridium perfringens in an experimental model of poultry necrotic enteritis. Vet Microbiol 142:323–328

    Article  Google Scholar 

  • Diarra MS, Malouin F (2014) Antibiotics in Canadian poultry productions and anticipated alternatives. Front Microbiol 5:282

    Article  Google Scholar 

  • Eid S, El Atfehy NM, Amer F, Tolba HN, Hamed RI (2020) Prevention of necrotic enteritis in broiler chickens by prebiotics and probiotics VS control by antibiotics, in vivo study. Alex J Vet Sci 64

  • Finegold SM (1989) Mechanisms of resistance in anaerobes and new developments in testing. Diagn Microbiol Infect Dis 12:117–120

    Article  Google Scholar 

  • El-Shibiny A, Scott A, Timms A, Metawea Y, Connerton P, Connerton I (2009) Application of a group II Campylobacter bacteriophage to reduce strains of Campylobacter jejuni and Campylobacter coli colonizing broiler chickens. J Food Prot 72:733–740

    Article  CAS  Google Scholar 

  • Gad W, Hauck R, Krüger M, Hafez H (2011) Determination of antibiotic sensitivities of Clostridium perfringens isolates from commercial turkeys in Germany in vitro. Europ PoultSci 75:80–83

    Google Scholar 

  • Gholamiandehkordi AR, Timbermont L, Lanckriet A, Broeck WVD, Pedersen K, Dewulf J, Pasmans F, Haesebrouck F, Ducatelle R, Immerseel FV (2007) Quantification of gut lesions in a subclinical necrotic enteritis model. Avian Pathol 36:375–382

    Article  Google Scholar 

  • Ha E, Son B, Ryu S (2018) Clostridium perfringens virulent bacteriophage CPS2 and its thermostable endolysin lysCPS2. Viruses 10:251

    Article  Google Scholar 

  • Hsu BB, Gibson TE, Yeliseyev V, Liu Q, Lyon L, Bry L, Silver PA, Gerber G.K (2019) Dynamic modulation of the gut microbiota and metabolome by bacteriophages in a mouse model. Cell host microbe 25, 803–814. e805

  • 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 Pathog 4:e26. https://doi.org/10.1371/journal.ppat.0040026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Keyburn AL, Sheedy SA, Ford ME, Williamson MM, Awad MM, Rood JI, Moore RJJI (2006) Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens. Infect Immun 74:6496–6500

    Article  CAS  Google Scholar 

  • Lau G, Sieo C, Tan W, Hair-Bejo M, Jalila A, Ho Y (2010) Efficacy of a bacteriophage isolated from chickens as a therapeutic agent for colibacillosis in broiler chickens. Poult Sci 89:2589–2596

    Article  CAS  Google Scholar 

  • M'Sadeq SA, Wu S, Swick RA, Choct M (2015) Towards the control of necrotic enteritis in broiler chickens with in-feed antibiotics phasing-out worldwide. Anim Nutr 1:1–11

    Article  Google Scholar 

  • Martin TG, Smyth JA (2009) Prevalence of netB among some clinical isolates of Clostridium perfringens from animals in the United States. Vet Microbiol 136:202–205

    Article  CAS  Google Scholar 

  • Merati R, Temim S, Mohamed AAA (2017) Identication and characterization of Clostridium perfringens isolated from necrotic enteritisin broiler chickens in Tiaret, Western Algeria. Kafkas Univ Vet Fak Derg 23:595–601

    Google Scholar 

  • Miller RW, Skinner J, Sulakvelidze A, Mathis GF, Hofacre CL (2010) Bacteriophage therapy for control of necrotic enteritis of broiler chickens experimentally infected with Clostridium perfringens. Avian Dis 54:33–40

    Article  Google Scholar 

  • Moore RJ (2016) Necrotic enteritis predisposing factors in broiler chickens. Avian Pathol 45:275–281

    Article  CAS  Google Scholar 

  • Nabil NM, Tawakol MM, Hassan HM (2018) Assessing the impact of bacteriophages in the treatment of Salmonella in broiler chickens. Infect Ecol Epidemiol 8:1539056

    PubMed  PubMed Central  Google Scholar 

  • National Research Council (1994) Nutrient requirements of poultry ninth revised edition National Academy Press, Washington DC

  • Oechslin F (2018) Resistance development to bacteriophages occurring during bacteriophage therapy. Viruses 10:351

    Article  Google Scholar 

  • Palliyeguru MCD, Rose S, Mackenzie A (2011) Effect of trypsin inhibitor activity in soya bean on growth performance, protein digestibility and incidence of sub-clinical necrotic enteritis in broiler chicken flocks. Br Poult Sci 52:359–367

    Article  CAS  Google Scholar 

  • Park JY, Kim S, Oh JY, Kim HR, Jang I, Lee HS, Kwon YK (2015) Characterization of Clostridium perfringens isolates obtained from 2010 to 2012 from chickens with necrotic enteritis in Korea. Poult Sci 94:1158–1164

    Article  CAS  Google Scholar 

  • Phothichaisri W, Ounjai P, Phetruen T, Janvilisri T, Khunrae P, Singhakaew S, Wangroongsarb P, Chankhamhaengdecha S (2018) Characterization of bacteriophages infecting clinical isolates of Clostridium difficile. Fronti Microbiol 9:1701

    Article  Google Scholar 

  • Redondo LM, Redondo EA, Delgado FO, La Sala LF, Fernandez Miyakawa ME (2016) An experimental reproduction of necrotic enteritis in broiler chickens. J Vet Sci Med 4:5

    Article  Google Scholar 

  • Richards PJ, Connerton PL, Connerton IF (2019) Phage biocontrol of Campylobacter jejuni in chickens does not produce collateral effects on the gut microbiota. Fronti Microbiol 10:476

    Article  Google Scholar 

  • Rood JI, Adams V, Lacey J, Lyras D, McClane BA, Melville SB, Moore RJ, Popoff MR, Sarker MR, Songer JGJA (2018) Expansion of the Clostridium perfringens toxin-based typing scheme. Anaerobe 53:5–10

    Article  CAS  Google Scholar 

  • Seal BS (2013) Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium1. Poult Sci 92:526–533

    Article  CAS  Google Scholar 

  • Seal BS, Fouts DE, Simmons M, Garrish JK, Kuntz RL, Woolsey R, Schegg KM, Kropinski AM, Ackermann HW, Siragusa GR (2011) Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions. Arch Virol 156:25–35

    Article  CAS  Google Scholar 

  • Tessari ENC, Cardoso ALSP, Kanashiro AMI, Stoppa GFZ, Luciano RL, de Castro AGM (2014) Analysis of the presence of Clostridium perfringens in feed and raw material used in poultry production. Food Nutr Sci 5:614–617

    Google Scholar 

  • Tizhe JQ, Bello M, Kabir J, Musa JA, Lamurde NJ (2015) Isolation and biochemical identification of Clostridium perfringens from raw beef sold in retail outlets in Zaria Metropolis, Nigeria. Int J Curr Microbiol Appl Sci 4:23–29

    CAS  Google Scholar 

  • Vinner GK, Vladisavljević GT, Clokie MR, Malik DJ (2017) Microencapsulation of Clostridium difficile specific bacteriophages using microfluidic glass capillary devices for colon delivery using pH triggered release. PLoS One 12:e0186239

    Article  Google Scholar 

  • Volozhantsev NV, Oakley BB, Morales CA, Verevkin VV, Bannov VA, Krasilnikova VM, Popova AV, Zhilenkov EL, Garrish JK, Schegg KM (2012) Molecular characterization of podoviral bacteriophages virulent for Clostridium perfringens and their comparison with members of the Picovirinae. PLoS One 7:e38283

    Article  CAS  Google Scholar 

  • Wernicki A, Nowaczek A, Urban-Chmiel R (2017) Bacteriophage therapy to combat bacterial infections in poultry. Virol J 14:179

    Article  Google Scholar 

  • Wilkie DC, Van Kessel AG, White LJ, Laarveld B, Drew MD (2005) Dietary amino acids affect intestinal Clostridium perfringens populations in broiler chickens. Can J Anim Sci 85:185–193

    Article  CAS  Google Scholar 

  • Yang WY, Lee YJ, Lu HY, Branton SL, Chou CH, Wang CJPs (2019) The netB-positive Clostridium perfringens in the experimental induction of necrotic enteritis with or without predisposing factors. Poult Sci 98: 5297–5306

  • Żbikowska K, Michalczuk M, Dolka B (2020) The use of bacteriophages in the poultry industry. Animals 10:872

    Article  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Ahmed Erfan for his help during the examination of the molecular toxinotypes of the selected C. perfringens isolate using conventional polymerase chain reaction.

Availability of data and material

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary information.

Code availability

Non-applicable.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author information

Authors and Affiliations

  1. Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Giza, 264, Egypt

    Reham A. Hosny, Ahmed F. Gaber & Hend K. Sorour

Authors
  1. Reham A. Hosny
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ahmed F. Gaber
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hend K. Sorour
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Reham A. Hosny and Hend K. Sorour isolated C. perfringens and C. perfringens phage. Reham A. Hosny, Ahmed F. Gaber, and Hend K. Sorour designed and carried out the experiment, performed laboratory and pathological examination, interpreted the results. Reham A. Hosny performed all statistical analyses in the manuscript. Reham A. Hosny, Ahmed F. Gaber, and Hend K Sorour wrote and revised the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Reham A. Hosny.

Ethics declarations

Ethical approval

All animal handling procedures were done according to an approved protocol [AHRI (23) 27/3/2019] by the Ethical Committee of the Animal Health Research Institute, Ministry of Agriculture, Giza, Egypt.

Conflict of interest/competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Fig 1

Toxinotyping of C. perfringens host strain using a polymerase chain reaction. Lane 1: Examined sample, Lane 2: C. perfringens positive control ATCC 12917, Lane 3: C. perfringens positive control ATCC 27324, Lane 4: C. perfringens positive control ATCC 3626, and lane 5: ladder 100–600 bp. (PNG 47 kb)

High resolution image (TIF 2642 kb)

Fig 2

The thermal stability of C. perfringens phage in different dilutions at different temperatures of 30 °C, 50 °C, 70 °C, and 90 °C following a 60-min incubation using the plaque assay test. The phage (109 PFU/ml) was stable to heat at 30 °C with a log titer of 9.9 PFU/ml, with only a slight reduction in log titer of 9.8 PFU/ml at 50 °C, beyond which they were undetectable. (PNG 24 kb)

High resolution image (TIF 5031 kb)

Fig 3

The pH stability of C. perfringens phage in different dilutions at different pH values of 2, 4, 8, and 10 using the plaque assay test. The phage (109 PFU/ml) retained viability across the different pH values in the plaque assay method with log titers of 9.7, 9.8, 9.9, and 9.7 PFU/ml at pH values of 2, 4, and 8, and 10, respectively. (PNG 54 kb)

High resolution image (TIF 6031 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hosny, R.A., Gaber, A.F. & Sorour, H.K. Bacteriophage mediated control of necrotic enteritis caused by C. perfringens in broiler chickens.. Vet Res Commun 45, 409–421 (2021). https://doi.org/10.1007/s11259-021-09821-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11259-021-09821-3

Keywords

Search

Navigation