Eimeria species and genetic background influence the serum protein profile of broilers with coccidiosis

Abstract

Background: Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria.

Methodology/principal findings: Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at P<0.05-0.01 (250 spots), P<0.01-0.001 (248 spots), and P<0.001 (314 spots) were excised and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified in 172 spots. A total of 46 different proteins were identified. Of the spots with a corresponding protein identification, 57 showed a main effect of coccidia infection and/or 2-way interaction of coccidia infection×broiler genetic line at P<0.001.

Conclusions/significance: Several of the metabolic enzymes identified in this study are potential candidates for early diagnostic markers of E. acervulina infection including malate dehydrogenase 2, NADH dehydrogenase 1 alpha subcomplex 9, and an ATP synthase. These proteins were detected only in Line A birds that were inoculated with E. acervulina. Results from this study provide a basic framework for future research aimed at uncovering the complex biochemical mechanisms involved in host response to Eimeria infection and in identifying molecular targets for diagnostic screening and development of alternative preventative and therapeutic methods.

Figure 1. Body weights at Day 21 for Line A and B birds infected with 1 of 3 Eimeria strains.

Birds were inoculated at day 15 with vehicle (control.), E. acervulina (EA), E. maxima (EM) or E. tenella (ET). A. = Line A, B. = Line B. Bars represent means ± pooled SEM. Asterisks represent difference from respective control group within genetic line (P<0.01).

Figure 2. Lesion scores at day 21 (6 d post-infection) for Line A and Line B broilers infected with E. acervulina, E. maxima or E. tenella.

Birds were inoculated at 15 d of age. Intestines were evaluated at day 21 and scored for lesions by a single expert . Scores ranged from 0 (no lesions) to 4 (numerous severe lesions).

Figure 3. Master image of gel stained with Flamingo™ fluorescent stain following 2-dimensional gel electrophoresis of chicken serum protein.

The first dimension was carried out using pH 3–10 immobilized pH gradient strips and the second dimension was performed using 8–16% acrylamide gels. Annotated spots are those showing a significant effect of coccidia infection and/or genetic line×coccidia infection interaction at P<0.001 (Table 2).

Figure 4. Broiler genetic line×coccidia infection interactions (P<0.001) for complement component 3 and protease inhibitors.

Line A and B broiler chicks were either not infected (control) or were inoculated with 1 of 3 Eimeria species, E. acervulina (EA), E. maxima (EM) or E. tenella (ET) at 15 d post-hatch. At 21 d (6 d post-infection) serum was harvested from whole blood samples and used for 2D gel electrophoresis followed by MALDI TOF/TOF. Protein density was evaluated by densitometric analysis of spot intensity followed by ANOVA. Values represent mean density units ± SEM (N = 6).

Figure 5. Broiler genetic line×coccidia infection interactions (P<0.001) for iron-binding and blood-clotting proteins.

Line A and B broiler chicks were either not infected (control) or were inoculated with 1 of 3 Eimeria species, E. acervulina (EA), E. maxima (EM) or E. tenella (ET) at 15 d post-hatch. At 21 d (6 d post-infection) serum was harvested from whole blood samples and used for 2D gel electrophoresis followed by MALDI TOF/TOF. Protein density was evaluated by densitometric analysis of spot intensity followed by ANOVA. Values represent mean density units ± SEM (N = 6).

Figure 6. Broiler genetic line×coccidia infection interactions (P<0.001) for metabolic and oxidative-stress related proteins.

Line A and B broiler chicks were either not infected (control) or were inoculated with 1 of 3 Eimeria species, E. acervulina (EA), E. maxima (EM) or E. tenella (ET) at 15 d post-hatch. At 21 d (6 d post-infection) serum was harvested from whole blood samples and used for 2D gel electrophoresis followed by MALDI TOF/TOF. Protein density was evaluated by densitometric analysis of spot intensity followed by ANOVA. Values represent mean density units ± SEM (N = 6).

Figure 7. Broiler genetic line×coccidia infection interactions (P<0.001) for cargo and structural proteins.

Line A and B broiler chicks were either not infected (control) or were inoculated with 1 of 3 Eimeria species, E. acervulina (EA), E. maxima (EM) or E. tenella (ET) at 15 d post-hatch. At 21 d (6 d post-infection) serum was harvested from whole blood samples and used for 2D gel electrophoresis followed by MALDI TOF/TOF. Protein density was evaluated by densitometric analysis of spot intensity followed by ANOVA. Values represent mean density units ± SEM (N = 6).

Figure 8. Experimental design and workflow.

Line A and B chicks were inoculated at day 15 with E. acervulina (EA), E. maxima (EM) or E. tenella (ET), or the vehicle (control; C). Blood was collected and lesion scoring was performed on the small intestine. Serum samples were depleted of IgG and albumin and composited. Two-dimensional electrophoresis was perfomed and gels were stained with Flamingo stain. Protein spot density was analyzed and spots showing a significant effect of coccidia strain and/or broiler genetic line at P<0.05−0.01 (250 spots), P<0.01−0.001 (248 spots), and P<0.001 (314 spots) were excised and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.