Temporal Changes in Jejunal and Ileal Microbiota of Broiler Chickens with Clinical Coccidiosis (Eimeria maxima)

Abstract

Coccidiosis in broiler chickens continues to be a major disease of the gastrointestinal tract, causing economic losses to the poultry industry worldwide. The goal of this study was to generate a symptomatic Eimeria maxima (1000 oocysts) infection to determine its effect on the luminal and mucosal microbiota populations (L and M) in the jejunum and ileum (J and IL). Samples were taken from day 0 to 14 post-infection, and sequencing of 16S rRNA was performed using Illumina technology. Infected birds had significantly (p < 0.0001) lower body weight gain (BWG), higher feed conversion ratio (FCR) (p = 0.0015), increased crypt depth, and decreased villus height (p < 0.05). The significant differences in alpha and beta diversity were observed primarily at height of infection (D7). Analysis of taxonomy indicated that J-L and M were dominated by Lactobacillus, and in IL-M, changeover from Candidatus Arthromitus to Lactobacillus as the major taxon was observed, which occurred quicky in infected animals. LEfSe analysis found that in the J-M of infected chickens, Lactobacillus was significantly more abundant in infected (IF) chickens. These findings show that E. maxima infection affects the microbiota of the small intestine in a time-dependent manner, with different effects on the luminal and mucosal populations.

Keywords: 16S rRNA sequencing; Eimeria maxima; broilers; coccidiosis; gut morphology; microbiota.

Figure 1

Concentration of plasma carotenoids in control (C) and Eimeria maxima (IF)-infected chickens (day PI, day post-infection; mean ± SE).

Figure 2

Crypt elongation following infection with Eimeria maxima. Expression of Olfm4 mRNA by in situ hybridization in the jejunum of broiler chickens that were infected at 21 d of age with either 1000 E. maxima oocysts (IF) or sham infected with sterile water (C) and sampled at 0, 3, 5, 7, 10, and 14 d post-infection (PI). All tissues were counterstained with 50% hematoxylin. Images were captured at 40× magnification (n = 4). (A) Crypt depth and (B) villus height was measured on jejunal sections stained for Olfm4 by in situ hybridization. Measures were analyzed by infection status using the nonparametric Welch’s one-way test. Significances (p < 0.05) are indicated by asterisks (*).

Figure 3

Effect of Eimeria maxima infection at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on the alpha diversity indices (A) Shannon’s entropy and (B) Faith’s PD of the mucosal bacterial populations of the jejunum (J-M). Non-infected birds = C, infected birds = IF. Significant (p < 0.05) differences are indicated by asterisks (*).

Figure 4

Effect of Eimeria maxima infection at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on the alpha diversity indices (A) Shannon entropy, (B) observed features, Faith’s PD (C), and Evenness (D) of the luminal bacterial populations of the ileum (IL-C). Non-infected birds = C, infected birds = IF. Significant (p < 0.05) differences are indicated by asterisks (*).

Figure 5

Effect of Eimeria maxima at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on the beta diversity of jejunal luminal (A) (J-C) and jejunal mucosa (B) (J-M) bacterial populations using the principal coordinate analysis (PcoA) based on the weighted (A) and unweighted UniFrac (B) distances between groups. Non-infected birds = C, infected birds = IF. Significant (p  <  0.05) differences are indicated by asterisks (*).

Figure 6

Effect of Eimeria maxima at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on the beta diversity of ileal luminal (A,B) (IL-C) and ileal mucosa (C) (IL-M) bacterial populations using the principal coordinate analysis (PcoA) based on the weighted (A,C) and unweighted UniFrac (B) distances between groups. Non-infected birds = C, infected birds = IF. Significant (p < 0.05) differences are indicated by asterisks (*).

Figure 7

Effect of Eimeria maxima at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on relative bacterial abundance (%) at the genus level in the (A) jejunal lumen (J-C) and (B) jejunal mucosa (J-M). Non-infected birds = C, infected birds = IF.

Figure 8

Effect of Eimeria maxima at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on relative bacterial abundance (%) at the genus level in the (A) ileal lumen (IL-C) and (B) ileal mucosa (IL-M). Non-infected birds = C, infected birds = IF.

Figure 9

Effect of Eimeria maxima on differentially abundant bacterial genera as determined by linear discriminant analysis (LDA) effect size (LEfSe) analysis in jejunal lumen (J-C, (A)) and mucosal (J-M, (B)) bacterial populations. C—uninfected chickens, IF—infected chickens.

Figure 10

Effect of Eimeria maxima on differentially abundant bacterial genera as determined by linear discriminant analysis (LDA) effect size (LEfSe) analysis in ileal lumen (IL-C, (A)) and mucosal (IL-M, (B)) bacterial populations. C—uninfected chickens, IF—infected chickens.

Figure 11

Effect of Eimeria maxima infection on mean proportion (%) of predicted MetaCyc pathways (up to top 21 pathways shown) in the jejunal luminal (J-C) (A) and jejunal mucosal (J-M) (B) populations.

Figure 12

Effect of Eimeria maxima infection on the mean proportion (%) of predicted MetaCyc pathways (up to top 21 pathways shown) in the ileal luminal (IL-C) (A) and ileal mucosal (IL-M) (B) populations.