Parasitology Research

, Volume 112, Issue 6, pp 2299–2306 | Cite as

Embryonated chicken eggs as an alternative model for mixed Clostridium perfringens and Eimeria tenella infection in chickens

  • Alaa Aldin Alnassan
  • Awad Ali Shehata
  • Marianne Kotsch
  • Matthias Lendner
  • Arwid Daugschies
  • Berit Bangoura
Original Paper


The chorioallantoic membrane (CAM) of chicken embryo eggs is a suitable model for viral and bacterial infections. In the present study, a new approach for testing the pathogenesis and virulence of Clostridium perfringens and Eimeria tenella dual infections as a model using the CAM of embryonated chicken eggs was developed. For this purpose, 24 specific pathogen-free (SPF) embryonated chicken eggs were divided into four groups (n = 6) and designated group E, group CP, group CPE, and NC. Sporozoites of E. tenella (20,000 sporozoites) were inoculated into 10-day-old embryonated SPF chicken eggs (groups E and CPE) via allantoic sac route. At 15-day-old, eggs of groups CP and CPE were infected with 104 cfu C. perfringens via the same route. Assessment of pathogenicity was assessed using gross and histopathological lesions. Embryo mortality reached 17 % after mono-infection with C. perfringens and/or E. tenella and 50 % in the mixed-infected group. Lesions in the CAMs were most numerous and most severe in co-infected eggs (group CPE), reaching the maximum score of 3 in 50 % of the inoculated eggs (P < 0.01). In Eimeria spp.-infected eggs (group E), lesions of score were between 1 and 2. Mono-infection with C. perfringens did not lead to a significant occurrence of lesions. Histopathological investigations of the CAM revealed clusters of Gram-positive bacteria, infiltration with leukocytes, lymphocytes, and developmental stages of E. tenella in the co-infected group. These data suggest that embryonated eggs could be an in ovo model for studying the pathogenesis of mixed infection with Eimeria and C. perfringens.



The authors would like to thank the Institute of Animal Hygiene and Veterinary Public Health, University Leipzig, and Dr. Dörte Lüschow from Institute of Poultry Diseases, Free University of Berlin, for technical support.


  1. Adam R, Mussa S, Lindemann D, Oelschlaeger TA, Deadman M, Ferguson DJ, Moxon R, Schroten H (2002) The avian chorioallantoic membrane in ovo—a useful model for bacterial invasion assays. Int J Med Microbiol 292:267–275PubMedCrossRefGoogle Scholar
  2. Al-Sheikhly F, Al-Saieg A (1980) Role of coccidia in the occurrence of necrotic enteritis of chickens. Avian Dis 24:324–333PubMedCrossRefGoogle Scholar
  3. Baums CG, Schotte U, Amtsberg G, Goethe R (2004) Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet Microbiol 100:11–16PubMedCrossRefGoogle Scholar
  4. Hafeez MA, Akhtar M, Hussain I (2006) Protective effect of egg-propagated Eimeria tenella (local isolates) gametocytes as vaccine(s) against mixed species of coccidia in chickens. Parasitol Res 98:539–544PubMedCrossRefGoogle Scholar
  5. Hodgson JN (1970) Coccidiosis: oocyst counting technique for coccidiostat evaluation. Exp Parasitol 28:99–102PubMedCrossRefGoogle Scholar
  6. Hofmann J, Raether W (1990) Improved techniques for the in vitro cultivation of Eimeria tenella in primary chick kidney cells. Parasitol Res 76:479–486PubMedCrossRefGoogle Scholar
  7. Hong YH, Lillehoj HS, Lee SH, Dalloul RA, Lillehoj EP (2006) Analysis of chicken cytokine and chemokine gene expression following Eimeria acervulina and Eimeria tenella infections. Vet Immunol Immunopathol 114:209–223PubMedCrossRefGoogle Scholar
  8. Jacobsen ID, Grosse K, Slesiona S, Hube B, Berndt A, Brock M (2010) Embryonated eggs as an alternative infection model to investigate Aspergillus fumigatus virulence. Infect Immun 78:2995–3006PubMedCrossRefGoogle Scholar
  9. Janse EM, Jeurissen SH (1991) Ontogeny and function of two non-lymphoid cell populations in the chicken embryo. Immunobiology 182:472–481PubMedCrossRefGoogle Scholar
  10. Jiang L, Zhao Q, Zhu S, Han H, Dong H, Huang B (2012) Establishment of Eimeria tenella (local isolate) in chicken embryos. Parasite 19:285–289PubMedCrossRefGoogle Scholar
  11. Keyburn AL, Boyce JD, Vaz P, Bannam TL, Ford ME, Parker D, Di Rubbo A, Julian R, Robert JM (2008) NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS 4:1–11CrossRefGoogle Scholar
  12. Long PL, Millard BJ (1973) Eimeria infection of chicken embryos the effect of known anticoccidial drugs against E. tenella and E. mivati. Avian Pathol 2:111–125PubMedGoogle Scholar
  13. Lovland A, Kaldhusdal M (2001) Severely impaired production performance in broiler flocks with high incidence of Clostridium perfringens-associated hepatitis. Avian Pathol 30:73–81PubMedCrossRefGoogle Scholar
  14. Montgomery D, Jones S, Boyle R, LuoY BA (2005) The embryo lethality of Escherichia coli isolates and its relationship to various in vitro attributes. Avian Dis 49:63–69PubMedCrossRefGoogle Scholar
  15. Oblinger JL, Koburger JA (1975) Understanding and teaching the most probable number technique. J Milk Food Technol 38:540–545Google Scholar
  16. Park SS, Lillehoj HS, Allen PC, Park DW, FitzCoy S, Bautista DA, Lillehoje EP (2008) Immunopathology and cytokine responses in broiler chickens coinfected with Eimeria maxima and Clostridium perfringens with the use of an animal model of necrotic enteritis. Avian Dis 52:14–22PubMedCrossRefGoogle Scholar
  17. Raether W, Hofmann J, Uphoff M (1995) In: Eckert J, Braun R, Shirley MW (eds) Biotechnology guidelines on techniques in coccidiosis research. The European Communities, Luxembourg, pp 80–81Google Scholar
  18. Schmatz DM, Crane MS, Murray PK (1984) Purification of Eimeria sporozoites by DE-52 anion exchange chromatography. J Protozool 31:181–183PubMedGoogle Scholar
  19. Su Y, Fei A, Tsai F (2003) Differential diagnosis of five avian Eimeria species by polymerase chain reaction using primers derived from the internal transcribed spacer 1 (ITS-1) sequence. Vet Parasitol 117:221–227PubMedCrossRefGoogle Scholar
  20. Tierney J, Mulcahy G (2003) Comparative development of Eimeria tenella (Apicomplexa) in host cells in vitro. Parasitol Res 90:301–304PubMedCrossRefGoogle Scholar
  21. Tomley F (1997) Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites. Method San Diego Calif 13:171–176CrossRefGoogle Scholar
  22. Van der Sluis W (2000) Clostridial enteritis is an often underestimated problem. World Poult 16:42–43Google Scholar
  23. Van Immerseel F, Rood JI, Moore RJ, Titball RW (2009) Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens. Trends Microbiol 17:32–36PubMedCrossRefGoogle Scholar
  24. Vargas A, Zeisser-Labouèbe M, Lange N, Gurny R, Delie F (2007) The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems. Adv Drug Deliv Rev 59:1162–1176PubMedCrossRefGoogle Scholar
  25. Williams RB (2005) Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathol 34:159–180PubMedCrossRefGoogle Scholar
  26. Williams RB, Marshall RN, La Ragione RM, Catchpole J (2003) A new method for the experimental production of necrotic enteritis and its use for studies on the relationships between necrotic enteritis, coccidiosis and anticoccidial vaccination of chickens. Parasitol Res 90:19–26PubMedGoogle Scholar
  27. Wu SB, Rodgers N, Choct M (2010) Optimized necrotic enteritis model producing clinical and subclinical infection of Clostridium perfringens in broiler chickens. Avian Dis 54:1058–1065PubMedCrossRefGoogle Scholar
  28. Xie MQ, Fukata T, Gilbert JM, McDougald LR (1991) Evaluation of anticoccidial drugs in chicken embryos. Parasitol Res 77:595–599PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Alaa Aldin Alnassan
    • 1
  • Awad Ali Shehata
    • 2
    • 3
  • Marianne Kotsch
    • 2
  • Matthias Lendner
    • 1
  • Arwid Daugschies
    • 1
  • Berit Bangoura
    • 1
    • 4
  1. 1.Institute of Parasitology, Faculty of Veterinary Medicine, Centre for Infectious DiseasesUniversity of LeipzigLeipzigGermany
  2. 2.Institute for Bacteriology and Mycology, Faculty of Veterinary Medicine, Centre for Infectious DiseasesUniversity of LeipzigLeipzigGermany
  3. 3.Avian and Rabbit Diseases Department, Faculty of Veterinary MedicineMinoufiya UniversitySadat CityEgypt
  4. 4.Institute of Parasitology, Centre for Infectious Diseases, Faculty of Veterinary MedicineUniversity of LeipzigLeipzigGermany

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