Understanding the Respiratory Microbiome of Poultry in Ohio: Turkeys and Chicken Layers. John Ngunjiri

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1 Understanding the Respiratory Microbiome of Poultry in Ohio: Turkeys and Chicken Layers John Ngunjiri

2 Outline Achievements 2015/2016 commercial layer chicken study Comparison of chicken & turkey respiratory microbiome Pilot virus infection studies in SPF turkeys Efforts in virome sample processing Ongoing and future work

3 Outline Achievements 2015/2016 commercial layer chicken study Comparison of chicken & turkey respiratory microbiome Pilot virus infection studies in SPF turkeys Efforts in virome sample processing Ongoing and future work

4 Achievements in 2015/2016 vlongitudinal sampling of commercial flocks (November 2015 November 2016) Turkeys Chicken layers vcollected >1000 samples: trachea, sinus, gut, and serum vprocessed turkey samples v16s sequencing and analysis of turkey samples vpreparing to write the manuscript on turkey respiratory microbiome

5 Achievements in 2016/2017 vlongitudinal sampling of SPF turkeys Research flock at FAHRP facility vcommercial chicken samples from 2015/2016 study Sample processing 16S sequencing Bioinformatics analysis Serological tests vpilot respiratory virus infection studies in turkeys Avian influenza virus Swine influenza virus

6 Longitudinal study of SPF turkeys vturkeys were hatched and raised in our facility following the facility s rearing SOP Floor: shavings (0-5 wks), no shavings (6-16 wks) No vaccines or antibiotics No NDV, REO, HEV, FLU, B. avium, MS, MG, MM v Sampling has been completed 0, 1, 2, 3, 5, 8, 12,16 weeks post-hatch Trachea, sinus, ileum, cecum, shavings, floor swabs vmost of the samples have been processed vnext steps Sequence, analyze, compare with commercial flock data

7 Outline Achievements 2015/2016 commercial layer chicken study Comparison of chicken & turkey respiratory microbiome Pilot virus infection studies in SPF turkeys Efforts in virome sample processing Ongoing and future work

and trachea taxa profiles were different Community structure

8 Composition & dynamics of respiratory microbiome SINUS TRACHEA Brood Before laying Laying Relative abundance Sinus and trachea taxa profiles were different Community structure changed with age Mycoplasma infection in trachea at wks 12, 25, 36, 51

9 Immunological status of chickens sampled for respiratory microbiome Antibody Titers AEV IBV IBDV Antibody Titers REOVIRUS % positive M. GALLISEPTICUM M. SYNOVIAE Age in Weeks IBV, AEV, IBDV, and MG antibodies were due to vaccination Reovirus and mycoplasma antibodies were due to infection

10 Outline Achievements 2015/2016 commercial layer chicken study Comparison of chicken & turkey respiratory microbiome Pilot virus infection studies in SPF turkeys Efforts in virome sample processing Ongoing and future work

$Chicken Turkey Unifrac distance$ comparisons Significant

11 SINUS Beta-diversity TRACHEA Chicken Turkey Unifrac distance comparisons Significant differences diversity between bird types

chickens Staphylococcus sciuri in turkeys vabundance >50%

12 Core microbiome Chicken Sinus Turkey Sinus Relative Abundance vdominant core microbiome Lactobacilli in chickens Staphylococcus sciuri in turkeys vabundance >50% of total bacteria in turkeys <50% of total bacteria in older chickens

13 Baseline microbiome summary Differences were found in taxonomic profiles of sinus and trachea Age had significant influence on bacterial diversity Effect of infection or immunological status of birds on respiratory microbiome need further investigation Bird type has significant influence on diversity and dynamics of core microbiome

14 Outline Achievements 2015/2016 commercial layer chicken study Comparison of chicken & turkey respiratory microbiome Pilot virus infection studies in SPF turkeys Efforts in virome sample processing Ongoing and future work

15 Experimental Design 2 weeks old SPF poults Influenza viruses Avian A/CK/PA/13609/93 (H5N2) Swine A/TK/OH/313053/04 (H3N2) Infected with 10 6 EID 50 /0.2ml of virus per bird Euthanized at 5 dpi for sample collection

16 Virus infection vs turkey microbiome SINUS H5N2 Control Control H3N2 Observed OTUs * TRACHEA Observed OTUs *

17 Outline Achievements 2015/2016 commercial layer chicken study Comparison of chicken & turkey respiratory microbiome Pilot virus infection studies in SPF turkeys Efforts in virome sample processing Ongoing and future work

18 Virome sampling and sample processing Sample collection Sinus & Trachea was supernatants, tissue homogenates Need a sample processing protocol PEG precipitation protocol by Shah et al using adeno and influenza virus stocks Similar rates of precipitation for both viruses Ultracentrifugation significant loss of virus Will test whether PEG can remove rrna without ultracentrifugation

19 Outline Achievements 2015/2016 commercial layer chicken study Comparison of chicken & turkey respiratory microbiome Pilot virus infection studies in SPF turkeys Efforts in virome sample processing Ongoing and future work

20 Ongoing work SPF chicken study to compare upper and lower respiratory tract microbiome More analysis of 2015/2016 commercial chicken study Exploring methods for processing, sequencing, and analyzing virome samples Future work Longitudinal sampling of SPF chickens Virus infection studies in SPF and commercial birds Cage-free and organic flocks

Acknowledgments Lee Lab @ OSU Dr. Chang-Won Lee Dr.

21 Acknowledgments Lee OSU Dr. Chang-Won Lee Dr. John Ngunjiri Michael Abundo Hyesun Jang Dr. Mohamed Elaish Mahesh KC Amir Ghorbani Dr. Juliette Hanson and animal welfare personnel Saranga Wijeratne - MCIC Johnson UMN Dr. Timothy Johnson Dr. Bonnie Youmans Grant no

22 ADDITIONAL SLIDES

23 Diversity of chicken respiratory microbiome SINUS TRACHEA Observed OTUs PCoA Age was influential on beta-diversity Boxplots High species richness in sinus at 51 wks

24 Diversity of chicken respiratory microbiome SINUS TRACHEA PCoA Age was influential on beta-diversity

Lactobacilli in chickens vabundance <50% of total bacteria in

25 Core microbiome Chicken Trachea Turkey Trachea Relative Abundance vdominant core microbiome Burkholderia in turkeys Burkholderia and Lactobacilli in chickens vabundance <50% of total bacteria in turkeys at 5, 8, 12, 16 weeks <50% of total bacteria in chickens at 36, 51 weeks

26 H5N2 virus infection vs turkey microbiome SINUS TRACHEA Staphylococcus Streptococcus Enterobacteriaceae Pseudomonas Burkhoderia Enterococcus Lactobacillus Observed OTUs * H5N2 Control H5N2 Control

27 H3N2 virus infection vs turkey microbiome SINUS TRACHEA Micrococcaceae Staphylococcus Ruminococcus Burkholderia Lactobacillus Observed OTUs Control H3N2 Control * H3N2

28 2015/2016 commercial layer chicken study VACCINES & ANTIBIOTICS schedule Age in Weeks Vaccine/antibiotic ST IBDV IBDV IBDV Pox ST NDV LT IBV MG BMD antibiotic

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