Development of the Intestinal RNA Virus Community of Healthy Broiler Chickens

Abstract

Several RNA viruses such as astrovirus, rotavirus, reovirus and parvovirus have been detected in both healthy and diseased commercial poultry flocks. The aim of this study was to characterize (a) the development of the RNA viral community in the small intestines of healthy broiler chickens from hatch through 6 weeks of age (market age) and (b) the contribution of the breeder source vs. bird age in development of the community structure. Intestinal tissue samples were harvested from breeders and their progeny, processed for viral RNA extraction and sequenced using Illumina Hiseq sequencing technology resulting in 100 bp PE reads. The results from this study indicated that the breeder source influenced the RNA viral community only at hatch but later environment i.e. bird age had the more significant effect. The most abundant RNA viral family detected at 2, 4 and 6 weeks of age was Astroviridae, which decreased in abundance with age while the abundance of Picornaviridae increased with age.

Fig 1. Brief schematic representation of the steps involved in taxonomy mapping after the blastn.

Scripts 1, 2 and 3 were custom developed python scripts.

Fig 2. Rarefaction curves for all samples.

The curves for each sample were generated by varying subsampling level with intervals of 10,000 sequences except for hatch (H) samples which used intervals of 1,000 sequences. The Y-axis represents the number of genera and the X-axis represents the number of sequences.

Fig 3. Representation of alpha diversity indices.

(A) Number of observed genera (Sobs) for each group (B) Estimated richness (Chao) for each group (C) Diversity (Invsimpson) for each group (D) Two-way ANOVA on the Invsimpson indices where the Y-axis represents the Invsimpson index and the X-axis represents the age group. These alpha diversity indices were calculated on 137,668 randomly sampled sequences from H group and 270,349 randomly sampled sequences from the remaining groups.

Fig 4. Ordination analysis.

Non-metric multidimensional scaling plots to visualize the community distances calculated using Bray-Curtis dissimilarity index on (A) 137,668 sequences sub-sampled from each sample and (B) 270,349 sequences sub-sampled from each sample. The color legends are A: Green, H: Turquoise, 2W: Yellow, 2W_C: Pink, 4W: Black, 4W_C: Brown, 6W: Blue, and 6W_C: Red.

Fig 5. Differential abundance analysis.

Differential abundance (q < 0.1) of RNA viral families in progeny birds from breeder A vs. breeder mix (which excluded A) over different ages (2, 4 and 6 weeks of age). The differential abundance was computed using the edgeR package. For each RNA viral family, the median values of all birds in each group are used for heat map representation. The abundance of RNA viral families in A and H samples is plotted only for comparison. The blue-gray-red color scale represents low to high abundance.

Fig 6. Distribution of abundant RNA viral families in all groups.

For each RNA viral family, the median values of all birds in each group is used for heat map representation. The blue-gray-red color scale represents low to high abundance.

Fig 7. Abundance profiles for each age group.

For each RNA viral family, the median values for all the birds in each age group, irrespective of the breeder source, were used to calculate the percentage and only RNA viral families > 1% abundance are used for pie chart representations.