Caecal Microbiota of Experimentally Campylobacter jejuni- Infected Chickens at Different Ages

Abstract

Campylobacter jejuni is the most common bacterial cause of foodborne zoonosis in the European Union. Infections are often linked to the consumption and handling of poultry meat. The aim of the present study was to investigate the caecal microbiota of birds infected with C. jejuni at different ages. Therefore, a total of 180 birds of the laying hybrid Lohmann Brown-Classic were housed in 12 subgroups of 15 animals each in three performed repetitions. Three birds per subgroup were experimentally infected with C. jejuni at an age of about 21 days and about 78 days (4.46 ± 0.35 log₁₀ CFU/bird). Twenty-one days after experimental infection, microbiome studies were performed on 72 caecal samples of dissected birds (three primary infected and three further birds/subgroup). Amplification within the hypervariable region V 4 of the 16S rRNA gene was performed and sequenced with the Illumina MiSeq platform. Statistical analyses were performed using SAS^® Enterprise Guide^® (version 7.1) and R (version 3.5.2). Both factors, the experimental replication (p < 0.001) and the chickens' age at infection (p < 0.001) contributed significantly to the differences in microbial composition of the caecal samples. The factor experimental replication explained 24% of the sample's variability, whereas the factor age at infection explained 14% thereof. Twelve of 32 families showed a significantly different count profile between the two age groups, whereby strongest differences were seen for seven families, among them the family Campylobacteraceae (adjusted p = 0.003). The strongest difference between age groups was seen for a bacterial species that is assigned to the genus Turicibacter which in turn belongs to the family Erysipelotrichaceae (adjusted p < 0.0001). Correlation analyses revealed a common relationship in both chicken ages at infection between the absolute abundance of Campylobacteraceae and Alcaligenaceae, which consists of the genus Parasutterella. In general, concentrations of particular volatile fatty acids (VFA) demonstrated a negative correlation to absolute abundance of Campylobacteraceae, whereby the strongest link was seen for n-butyrate (-0.51141; p < 0.0001). Despite performing consecutive repetitions, the factor experimental replication contributed more to the differences of microbial composition in comparison to the factor age at infection.

Keywords: 16S rRNA; Campylobacter jejuni; foodborne pathogen; gut microbiota; volatile fatty acids.

FIGURE 1

Schedule of the experiments with actions in chickens of different ages at infection (LBC-21/22, Lohmann Brown-Classic, infection at the age of 21/22 days; LBC-70/78, Lohmann Brown-Classic, infection at the age of 70/78 days).

FIGURE 2

Bray-Curtis dissimilarity-based principal coordinate analysis (PCoA) was performed on caecal samples collected in experiments 1–3. Each point represents a different bird infected at different age [LBC-21/22, Lohmann Brown-Classic, infection at the age of 21/22 days (open points); LBC-70/78, Lohmann Brown-Classic, infection at the age of 70/78 days (filled points)]; coloured lines connect birds of one experiment (Exp 1, Experiment 1; Exp 2, Experiment 2; Exp3, Experiment 3).

FIGURE 3

Alpha diversity in samples from caecal content depending on experiment and age at infection. Box-plots showing alpha diversity in samples using the Observed Species index, the Chao1 index and Shannon index (Exp1, Experiment 1; Exp2, Experiment 2; Exp3, Experiment 3; LBC-21/22, Lohmann Brown-Classic, infection at the age of 21/22 days; LBC-70/78, Lohmann Brown-Classic, infection at the age of 70/78 days).

FIGURE 4

Microbiota composition analysis using 16S rRNA sequencing in caecal contents of different aged chickens at time of infection. Relative abundance of the 11 most abundant OTUs belonging to bacterial families within different phyla in experiment 1 (A). Relative abundance of the 11 most abundant OTUs belonging to bacterial families within different phyla in experiment 2 (B). Relative abundance of the 11 most abundant OTUs belonging to bacterial families within different phyla in experiment 3 (C). LBC-21/22, Lohmann Brown-Classic, infection at the age of 21/22 days; LBC-70/78, Lohmann Brown-Classic, infection at the age of 70/78 days; NA, OTUs without genus-level taxonomic assignment.

FIGURE 5

Left: Volcano plot representing –log10 FDR-adjusted p-values versus log2 fold changes for all 216 OTUs. Right: log2 fold changes of 28 OTUs, selected with a criterion of FDR-adjusted p-values < 0.05 and absolute log2 fold change >2. OTUs are ordered according to p-values, with the OTU_3 having the smallest p-value. Confidence intervals were calculated according to Jung et al. (2011).