The caecal microbiota promotes the acute inflammatory response and the loss of the intestinal barrier integrity during severe Eimeria tenella infection

Abstract

Introduction: Coccidiosis, a disease caused by intestinal apicomplexan parasites Eimeria, is a threat to poultry production. Eimeria tenella is one of the most pathogenic species, frequently causing a high prevalence of opportunistic infections.

Objective: The objective of this study is to investigate the role of the microbiota in the pathogenesis of severe Eimeria tenella infection.

Methods: We have previously shown that microbiota can promote parasite development. To study the effect of the microbiota on the pathogenesis of this infection, we used an experimental condition (inoculum of 10 000 oocysts E. tenella INRAE) in which the parasite load is similar between germ-free and conventional broilers at 7 days post-infection (pi). Thirteen conventional and 24 germ-free chickens were infected. Among this latter group, 12 remained germ-free and 12 received a microbiota from conventional healthy chickens at 4 days pi. Caeca and spleens were collected at 7 days pi.

Results: Our results demonstrated caecal lesions and epithelium damage in conventional chickens at 7 days pi but not in germ-free infected chickens. Administration of conventional microbiota to germ-free chickens partially restored these deleterious effects. At day 7 pi, both infected conventional and germ-free chickens exhibited increased gene expression of inflammatory mediators, including IL15, IFNγ, TNFα and the anti-inflammatory mediator SOCS1, whereas the inflammatory mediators CXCLi2, CCL20, IL18, CSF1, NOS2, PTGS2, IL1β, IL6, the receptor CCR2, and the anti-inflammatory mediators TGFβ1 and IL10 were upregulated only in infected conventional chickens. Notably, the IL18, PTGS2 gene expression was significantly higher in the infected conventional group. Overall, the inflammatory response enhanced by the microbiota might be in part responsible for higher lesion scores. Epithelial tight junction protein gene expression analysis revealed a significant upregulation of CLDN1 with the infection and microbiota, indicating a potential loss of the intestinal barrier integrity.

Conclusion: These observations imply that, during E. tenella infection, the caecal microbiota could trigger an acute inflammatory response, resulting in a loss of intestinal integrity. Increase in bacterial translocation can then lead to the likelihood of opportunistic infections. Hence, modulating the microbiota may offer a promising strategy for improving poultry gut health and limiting caecal coccidiosis.

Keywords: Eimeria tenella; bacterial translocation; chicken; germ-free; inflammation; microbiota.

Figure 1

Microbiota plays a major role in the generation of caecal lesions generated by E. tenella infection. (A) Conventional and germ-free chickens (n ≥ 12/group) were orally infected with E. tenella with a fixed dose of inoculum (10 000 oocysts/animal) leading to similar parasite load in germ-free and conventional chickens. At day 4 pi, a part (12 chickens) of infected germ-free chickens received a microbiota from conventional and healthy three-week-old chickens. Lesions were assessed at day 7 pi. Statistical analysis was performed on lesion scores using a Kruskall-Wallis test with a Dunn’s multiple comparisons post-test. Conventional versus germ-free: ****P < 0.0001; germ-free versus germ-free + microbiota: ****P < 0.0001; conventional versus germ-free + microbiota: ns: non significant (B) Germ-free chickens were infected for 6 days with a high dose of inoculum (10 000 or 50 000 oocysts/animal; n ≥ 4). Lesion scores were evaluated by the method described by (Johnson and Reid, 1970) and assessed on each caecum (two per animal). Statistical analysis was performed on lesion scores using Mann and Whitney test. **P < 0.01.

Figure 2

Microbiota favours mucosal damages and increased thickness associated with E. tenella infection. (A) Mucosal damage was less frequently observed in germ-free E. tenella infected chickens compared to conventional infected chickens. Representative histopathological pictures of caeca. Compared to control non-infected animals, infected conventional, germ-free, and germ-free receiving intestinal microbiota animals displayed respectively a severe (ulcerated epithelium indicated into black arrowhead), mild, and marked mucosa thickening (double black arrow). Thickening of the mucosa and submucosa was mainly composed of severe mixed inflammatory cell infiltration (*). E. tenella (open arrowhead) were mainly observed within epithelial cells of the mucosa. Hemalun-Eosin-Saffran. Bar=100µm. (B) Evaluation of the percentage of animals with epithelium damage (black arrow head) for each infected group. (C) Measure of the mucosal thickness (double black arrow) was performed for each animal and are expressed as the median (n ≥ 6/group). Statistical analysis was performed using a Kruskall-Wallis test with a Dunn’s multiple comparisons post-test. *P < 0.05; **P < 0.01; ****P < 0.0001; ns: non significant.

Figure 3

Increase in the percentage of CD45 positive cells in the caecal tissues of infected chickens both germ-free and conventional animals compared to non-infected chickens. Caeca were collected at 7 days pi. Cells were isolated, stained with anti-CD45 antibody, assessed for cell viability (ZombieAqua™) and analysed by flow cytometry (A) as described in the materials and methods section. Data are expressed as the median (n ≥ 5/group for at least 2 experiments). Statistical analysis was performed using a Kruskall-Wallis test with a Dunn’s multiple comparisons post-test. ****P < 0.0001; ns: non significant. (B) Representative image of CD45⁺ cells recruitment in E. tenella infected conventional and germ-free chickens compared to non-infected chickens (SP8 confocal laser-scanning Leica microscope; magnification 63X; image: 2048×2048 pixels, Z stacks and mosaic merge; Leica Application Suite X software).

Figure 4

Genes associated to inflammation in germ-free infected chickens are different than the one in conventional infected chickens. (A) Principal component analysis (PCA) of RT-qPCR gene expression values from caeca of infected and non-infected chickens following presence or absence of microbiota. Each chicken represented as a dot in specific colour according to its group assignment. Dim 1 (dimension 1) explained 64.4 of the total data variation between animals and Dim 2 (dimension 2) a further 10.2 of the variation. Barycenters for each group are represented in large dots compared to individual values. PERMANOVA test confirmed a significant difference between non-infected and infected chicken clusters. No dissimilarity was observed in conventional and germ-free non-infected groups. At 7 days pi conventional and germ-free clusters were distinct. However germ-free + microbiota cluster was not different from conventional and germ-free infected chickens. (B) Z-score hierarchical clustering heatmap depending of median genes expression. Colours represent scaled median values of gene expression with blue for low and red for high values.

Figure 5

Detailed gene expression assessed by RT-qPCR. Data are expressed as the median. (n ≥ 10/group). Statistical analysis was performed using a Kruskall-Wallis test with a Dunn’s multiple comparisons post-test. **P < 0.01 *** P < 0.001 ****P < 0.0001; ns: non significant.

Figure 6

CLDN1 gene expression increase during infection by E. tenella is dependent on the microbiota. Gene expression was assessed by qPCR. CLDN1 gene expression was increased in conventional chickens at 7 days pi compared to germ-free infected chickens. CLDN2 gene expression was increased while OCLN gene expression was decreased in conventional chickens at 7 days pi compared to conventional non-infected chickens. However, these gene expressions were unchanged in conventional infected chickens compared to germ-free infected chickens due to high variance. Data are expressed as the median (n ≥ 10/group). Statistical analysis was performed using a Kruskall-Wallis test with a Dunn’s multiple comparisons post-test. *P < 0.05 **P < 0.01 *** P < 0.001 ****P < 0.0001; ns: non significant.

Figure 7

E. tenella infection leads to bacterial translocation. (A) Commensal enterobacteria were followed in the spleen of infected and non-infected chickens at 7 days pi (n ≥ 7/group). (B) APEC were administered to the animals at 1 day old. Chickens were infected with E. tenella at 14 days old. Translocation of bacteria in the spleen was performed at day 7 pi (n = 10). Data are expressed as the number of spleens positive or negative for bacteria.

Figure 8

Summary diagram describing the influence of the caecal microbiota on the development of inflammation and the loss of intestinal integrity at day 7 pi with E. tenella. Created with BioRender.com (JV25NHUMTD, FE257PUXU1, RV257PUXXN).