Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages

Abstract

The effect of dietary fat source (soy oil or a mixture of lard and tallow) and dietary supplementation with antibiotics (a combination of avilamycin at 10 mg kg of feed(-1) and salinomycin at 40 mg kg of feed(-1)) on the bacterial community in the ileum of broiler chickens at different ages (7, 14, 21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture. The bacterial origin of fragments in DGGE profiles was identified by sequencing. Bacterial enumeration results, together with PCR-DGGE profiles, showed that the composition of the microflora was age dependent and influenced by dietary fat source and antibiotic supplementation. An increased incidence of streptococci, enterobacteria, and Clostridium perfringens with age of the chickens was demonstrated. Lactobacilli and C. perfringens were the bacterial groups most strongly affected by the dietary treatments. Moreover, different strains (clonal variants of the alpha-toxin gene) of C. perfringens type A were detected in response to age, dietary fat source, and dietary supplementation with antibiotics.

FIG. 1.

PCR-DGGE profiles (30 to 55% denaturing gradient gel) generated from ileal template DNA from 7-day-old chickens, using primer pair HDA1-GC plus HDA2. Lanes 1 to 8 are from replicate pooled samples from 16 chickens fed the S⁻ diet, and lanes 9 to 16 are from replicate pooled samples from 16 chickens fed the S⁺ diet. Note the consistency of profiles detected in birds of the same age.

FIG. 2.

PCR-DGGE profiles (30 to 55% denaturing gradient gel) generated from representative ileal content pools, using primer pair HDA1-GC plus HDA2. Lanes: 1 to 4, samples from 7-day-old chickens fed the A⁻ diet (lane 1), the A⁺ diet (lane 2), the S⁻ diet (lane 3), and the S⁺ diet (lane 4); 5 to 8, samples from 14-day-old chickens fed the A⁻ diet (lane 5), the A⁺ diet (lane 6), the S⁻ diet (lane 7), and the S⁺ diet (lane 8); 9 to 12, samples from 21-day-old chickens fed the A⁻ diet (lane 9), the A⁺ diet (lane 10), the S⁻ diet (lane 11), and the S⁺ diet (lane 12); 13 to 16, samples from 35-day-old chickens fed the A⁻ diet (lane 13), the A⁺ diet (lane 14), the S⁻ diet (lane 15), and the S⁺ diet (lane 16). The fragments were allotted by sequence analysis to the following genera or species: fragment a, L. johnsonii; fragment b, L. crispatus; fragment c, L. salivarius; fragment d, Streptococcus spp.; fragment e, sequence similarity of 98% to an uncultured bacterium and 97% to L. reuteri; fragment f, C. perfringens.

FIG. 3.

Detection of lactobacilli in representative ileal content pools, using primer pair Lac1 plus Lac2-GC in a 30 to 45% gradient gel. Lanes: 1 to 4, samples from 7-day-old chickens fed the A⁻ diet (lane 1), the A⁺ diet (lane 2), the S⁻ diet (lane 3), and the S⁺ diet (lane 4); 5 to 8, samples from 14-day-old chickens fed the A⁻ diet (lane 5), the A⁺ diet (lane 6), the S⁻ diet (lane 7), and the S⁺ diet (lane 8); 9, identification ladder composed of sequences from reference strains of Lactobacillus species (a, L. plantarum; b, L. johnsonii; c, Weissella viridescens; d, L. gasseri; e, L. acidophilus; f, L. crispatus; g, L. salivarius; h, L. ruminis; i, L. rhamnosus; j, L. reuteri); 10 to 13, samples from 21-day-old chickens fed the A⁻ diet (lane 10), the A⁺ diet (lane 11), the S⁻ diet (lane 12), and the S⁺ diet (lane 13); 14 to 17, samples from 35-day-old chickens fed the A⁻ diet (lane 14), the A⁺ diet (lane 15), the S⁻ diet (lane 16), and the S⁺ diet (lane 17).

FIG. 4.

Detection of C. perfringens in ileal content pools, using primer pair Cpa1-GC plus Cpa2 in a 20 to 35% gradient gel. Lanes: 1, C. perfringens ATCC 13124^T; 2 to 5, samples from 7-day-old chickens fed the A⁻ diet (lane 2), the A⁺ diet (lane 3), the S⁻ diet (lane 4), and the S⁺ diet (lane 5); 6 to 9, samples from 14-day-old chickens fed the A⁻ diet (lane 6), the A⁺ diet (lane 7), the S⁻ diet (lane 8), and the S⁺ diet (lane 9); 10 to 13, samples from 21-day-old chickens fed the A⁻ diet (lane 10), the A⁺ diet (lane 11), the S⁻ diet (lane 12), and the S⁺ diet (lane 13); 14 to 17, samples from 35-day-old chickens fed the A⁻ diet (lane 14), the A⁺ diet (lane 15), the S⁻ diet (lane 16), and the S⁺ diet (lane 17); 18, C. perfringens ATCC 13124^T. Fragments a (AF477010), b (AF477008), c (AF475144), and d (AF477009) represent four different strains of C. perfringens type A.