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

Abstract

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 10 (4) 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.