Impact of Eimeria tenella Oocyst Dose on Parasite Replication, Lesion Score and Cytokine Transcription in the Caeca in Three Breeds of Commercial Layer Chickens

Abstract

Eimeria species parasites infect the gastrointestinal tract of chickens, causing disease and impacting on production. The poultry industry relies on anticoccidial drugs and live vaccines to control Eimeria and there is a need for novel, scalable alternatives. Understanding the outcomes of experimental infection in commercial chickens is valuable for assessment of novel interventions. We examined the impact of different infectious doses of Eimeria tenella (one low dose, three high doses) in three commercial layer chicken lines, evaluating lesion score, parasite replication and cytokine response in the caeca. Groups of eight to ten chickens were housed together and infected with 250, 4,000, 8,000 or 12,000 sporulated oocysts at 21 days of age. Five days post-infection caeca were assessed for lesions and to quantify parasite replication by qPCR and cytokine transcription by RT-qPCR. Comparison of the three high doses revealed no significant variation between them in observed lesions or parasite replication with all being significantly higher than the low dose infection. Transcription of IFN-γ and IL-10 increased in all infected chickens relative to unchallenged controls, with no significant differences associated with dose magnitude (p > 0.05). No significant differences were detected in lesion score, parasite replication or caecal cytokine expression between the three lines of chickens. We therefore propose 4,000 E. tenella oocysts is a sufficient dose to reliably induce lesions in commercial layer chickens, and that estimates of parasite replication can be derived by qPCR from these same birds. However, more accurate quantification of Eimeria replication requires a separate low dose challenge group. Optimisation of challenge dose in an appropriate chicken line is essential to maximize the value of in vivo efficacy studies. For coccidiosis, this approach can reduce the numbers of chickens required for statistically significant studies and reduce experimental severity.

Keywords: Eimeria tenella; chicken; dose; experimental optimisation; layer; lesion; qPCR.

Figure 1

Lesion scores and parasite replication in caecal tissue measured by qPCR at 5 days post E. tenella infection in three layer chicken lines. Graphs (A–C) show lesion scores in chickens challenged with E. tenella. Each point demonstrates caecal lesion score of an individual bird 5 days post-challenge. Graphs (D–F) show parasite replication assessed by qPCR in chickens challenged with E. tenella. Each point demonstrates normalized parasite genome copy number (E. tenella gene copy/chicken TBP gene copy) from individual chickens 5 days post-challenge. Lines show the mean and standard deviation of each group. (A,D) show Lohmann Brown chickens, (B,E) show Hy-line Brown chickens, (C,F) show Hy-line Silver Brown chickens. Significant differences between means (One-way ANOVA and Tukeys correction) are shown; compared with control group (a) and with 250 oocyst dose (b), *p < 0.05, **p < 0.01.

Figure 2

IFN-γ and IL-10 transcription in caecal tissue at 5 days post E. tenella infection in three layer chicken lines. Graphs (A–C) show relative normalized gene expression (ΔΔCq) of IFN-γ in caecal tissues from chickens challenged with E. tenella. Each point demonstrates transcription of IFN-γ, relative to zero, in the caeca 5 days post challenge. Graphs (D–F) show relative normalized gene expression (ΔΔCq) of IL-10 in caecal tissues from chickens challenged with E. tenella. Each point demonstrates transcription of IL-10, relative to zero, in the caeca 5 days post challenge. Lines show the mean and standard deviation of each group. (A,D) show Lohmann Brown chickens. (B,E) show Hy-line Brown chickens. (C,F) show Hy-line Silver Brown chickens. *One point in graph E is not displayed and had normalized gene expression above 1,500 (1,933.7).

Figure 3

Lesion scores and parasite replication in caecal tissue measured by qPCR at 6 days post E. tenella infection in Hy-line brown chickens used as controls in a subsequent vaccine study. Graph (A) shows lesion scores in chickens challenged with E. tenella. Each point indicates the caecal lesion score from an individual chicken 6 days post-challenge. Graph (B) shows parasite replication assessed by qPCR in chickens challenged with E. tenella. Each point indicates the normalized parasite genome copy number (E. tenella gene copy/chicken TBP gene copy) from individual chickens 6 days post-challenge. Lines show the mean and standard deviation of each group.