Characterizing the Respiratory Microbiome of Commercial Broilers on the Delmarva Peninsula

Transcription

1 Characterizing the Respiratory Microbiome of Commercial Broilers on the Delmarva Peninsula Kelly Mulholland 1,2, Calvin Keeler, Jr. 2, Sharon J. Keeler 2 and Monique Robinson 2 1 Center for Bioinformatics and Systems Biology 2 Department of Animal and Food Sciences

2 The Avian Respiratory Microbiome Microbiomes are complex environments containing bacteria, viruses, fungi/yeast, archaea and protozoa Disturbances of this environment by colonization of a new bacteria, virus, or fungi can result in disease The avian respiratory disease complex (RDC) is a complex multiagent syndrome Understanding the etiology of RDC requires quantification of the microbial composition of the avian respiratory tract in both healthy and sick flocks 2

3 Challenges with Determining the Avian Respiratory Virome Sampling the respiratory tract Managing dilute samples Developing avian-specific bioinformatics tools viral genome databases method for detecting and quantifying viruses Aggregating and presenting bacteria, bacteriophage, fungi/yeast and virus data 3

4 Challenges with Determining the Avian Respiratory Virome Sampling the respiratory tract Managing dilute samples Developing avian-specific bioinformatics tools viral genome databases method for detecting and quantifying viruses Aggregating and presenting bacteria, bacteriophage, fungi/yeast and virus data 4

5 Objectives 1. Develop an automated workflow for identifying viral community abundance within the avian respiratory microbiome with the use of metagenomics NGS data and avian-specific viral genome databases. 2. Use these tools to elucidate microbial changes occurring during the grow out of a healthy broiler flock. 5

6 Experimental Design A healthy, untreated flock of chickens were sampled at placement and again at weeks 1, 2, 3, 4, 5,6 and 7. Tracheal swabs were collected, DNA and RNA was isolated and libraries were created DNAseq (DNA viruses, bacteriophage and Yeast/Fungi), RNAseq (RNA viruses), and 16S rrna (Bacteria) data were collected at each time point. DNA and RNAseq was conducted using Illumina HiSeq NGS technology and processed using following viral workflow for virus detection and quantification 6

7 Quality and Decontamination Fastq Files Trim Sequences for Quality Trimmed Files Align reads to Host Genome Chicken Genome Sequencedependent alignment Bacteriophage DB UnMapped Reads Host-Specific Viral Genome DB UnMapped Reads Quantification and data analysis Mapped Reads Normalized abundance % relative abundance Coverage Alpha and Beta Diversity Pearson Correlation Coefficients Network Analysis Mapped Reads 7

8 Database Construction A robust, curated avian viral genome databases is critical to virome studies To resolve this issue, databases were constructed using viral reference genomes from NCBI and contain 1-4 families for every virus class Avian DNA viral genome database Avian RNA viral genome database ~35 viruses 8 families ~65 viruses 12 families 8

9 Avian DNA viral genome database Double Stranded SS/DS Single Stranded Enveloped Non- Enveloped Enveloped Non- Enveloped Poxviridae (3) Herpesviridae (6) Adenoviridae (14) Hepeviridae (1) Hepadnaviridae (1) Genomoviridae (3) Circoviridae (10) Parvoviridae (7) 9

10 Avian RNA viral genome RNA database Double Stranded Single Stranded Positive Negative Positive Segmented Segmented Non- Segmented Non- Segmented Non- Enveloped Enveloped Enveloped Non- Enveloped Enveloped Reoviridae e (6) Orthomyxoviridae Orthomyxoviridae (16) (16) Phenuiviridae Phenuiviridae (2) (2) Bornaviridae Bornaviridae (1) (1) Pneumoviridae Pneumoviridae (1) (1) Paramyxoviridae Paramyxoviridae (10) (10) Astroviridae Astroviridae (5) (5) Calciviridae Picornaviridae (13) (0) Picornaviridae Calciviridae (0) (13) Retroviridae Retroviridae (5) (5) Flaviviridae Flaviviridae (2) (2) Coronaviridae Coronaviridae (5) (5)

11 Sample Statistics Sample Total Reads After Trimming % Mapped % UnMapped Mapped to Chicken Chicken Viral Reads Week 0 DNA 46,586,608 46,568, Week 0 RNA 62,375,955 62,351, Week1 DNA 43,940,202 43,911, Week1 RNA 54,496,930 54,477, ,569 Week 2 DNA 33,471,831 33,442, Week 2 RNA 51,426,090 51,415, ,016 Week 3 DNA 45,131,953 45,108, Week 3 RNA 57,566,851 57,551, Week 4 DNA 44,621,969 44,600, Week 4 RNA 56,334,014 56,321, ,660 Week 5 DNA 38,630,592 38,590, Week 5 RNA 50,937,821 50,926, ,579 Week 6 DNA 45,915,721 45,900, ,634 Week 6 RNA 53,248,431 53,236, ,476 Week 7 DNA 41,215,207 41,196, Week 7 RNA 54,173,052 54,162, ,243 Total Raw Reads: ~780 Million Average % DNA UnMapped: 12.42% Average % RNA UnMapped: 46.56% 11

12 DNA/RNA Viruses Detected During Grow Out Viral Abundance * DNA RNA DNA RNA DNA RNA DNA RNA DNA RNA DNA RNA DNA RNA DNA RNA Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Turkey coronavirus Infectious bursal disease virus segment B Infectious bursal disease virus segment A Infectious bronchitis virus strain Georgia 1998 Vaccine Infectious bronchitis virus strain Arkansas DPI Chicken sicinivirus JSY Chicken gallivirus 1 isolate 518C Chicken astrovirus Avian orthoreovirus segment L1 Avian myelocytomatosis virus Avian leukosis virus Avian leukemia virus isolate SCDY1 Avian infectious bronchitis virus strain Mass 41 Avian infectious bronchitis virus Avian endogenous retrovirus EAV-HP Avian carcinoma virus Meleagrid herpesvirus 1 Gallid herpesvirus 3 Gallid herpesvirus 2 Gallid herpesvirus 1 Fowl adenovirus E Fowl adenovirus A Chicken anemia virus Avian gyrovirus 2 12

13 Viral Families Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 DNA RNA Poxviridae Herpesviridae Adenoviridae Hepeviridae Hepadnaviridae Genomiviridae Circoviridae Parvoviridae Reoviridae Orthomyxoviridae Phenuiviridae Bornaviridae Pneumoviridae Paramyxoviridae Astroviridae Picornoviridae Retroviridae Flaviviridae Coronaviridae 13

14 Quantifying Viral Abundance of Each Sample Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Gallid Alphaherpesvirus DNA Viruses Meleagrid Alphaherpesvirus 1 Gallid Alphaherpesvirus 2&3 Avian Gyrovirus Fowl Adenovirus RNA Viruses Chicken Astrovirus Chicken Sicinivirus JSY Avian Carcinoma Virus Avian Infectious Bronchitis Virus Infectious Bursal Disease Virus Avian Endogenous Retrovirus

15 Viral Alpha Diversity Alpha diversity was measured for viruses utilizing the Shannon Diversity Index: 15

16 Bacteria - 16s rrna Week WEEK0 1 Week 7 Pseudomonadacea e 18% Corynebacteriaceae 8% Brevibacteriaceae 13% Corynebacteriaceae 23% Lactobacillales 9% Streptococcaceae 11% Bacillaceae 9% Staphylococcaceae 7% Bacillaceae 4% Dermabacteraceae Dermabacteraceae 11% 11% Flavobacteriaceae 10% Pasteurellaceae 38% Ruminococcaceae 1% Lactobacillales 15% Bacillaceae 2% Streptococcaceae 2% Brevibacteriaceae 3% Dermabacteraceae 2% Bacillaceae 1% Staphylococcaceae 13% 16

17 Bacterial Alpha Diversity Week 0 Week 1 Week 3 Week 4 Week 5 Week 7 Alpha diversity was measured for bacteria utilizing the Shannon Diversity Index: 17

18 Bacteria Bacteriophage Correlations Pearson Correlation Coefficient 18

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20 Conclusions/Future Directions Developed avian-specific bioinformatics tools and applied them to detect the microbial shift in the respiratory tract of a healthy broiler flock during grow out Identify microbial shift between a healthy flock and a flock exhibiting respiratory disease complex. Conduct a comparison of identified viruses, bacteria, fungi and phage. Compare respiratory microbiota detected in a healthy flock to gastrointestinal microbiota Implement the present workflow and databases into a user-friendly interface to facilitate future investigators interested in rapidly characterizing and quantifying the viral component of a microbiome 20

21 Thank You 21

22 Quantifying Abundance of Each Virus Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Gallid Alphaherpesvirus DNA Viruses Meleagrid Alphaherpesvirus 1 Gallid Alphaherpesvirus 2&3 Avian Gyrovirus Fowl Adenovirus Chicken Astrovirus RNA Viruses Chicken Sicinivirus JSY Avian Carcinoma Virus Avian Infectious Bronchitis Virus Infectious Bursal Disease Virus Avian Endogenous Retrovirus