bioRxiv preprint doi: https://doi.org/10.1101/2020.02.19.957092; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 RESPIRATORY AND GUT MICROBIOTA IN COMMERCIAL TURKEY FLOCKS WITH 2 DISPARATE WEIGHT GAIN TRAJECTORIES DISPLAY DIFFERENTIAL 3 COMPOSITIONAL DYNAMICS 4 5 Kara J.M. Taylor1‡, John M. Ngunjiri1‡, Michael C. Abundo1,2, Hyesun Jang1,2, Mohamed 6 Elaish1, Amir Ghorbani1,2, Mahesh KC1,2, Bonnie P. Weber3, Timothy J. Johnson3,4, Chang-Won 1,2* 7 Lee 8 9 1Food Animal Health Research Program, Ohio Agricultural Research and Development Center, 10 The Ohio State University, Wooster, Ohio, United States of America 11 2Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio 12 State University, Columbus, Ohio, United States of America 13 3Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, 14 Minnesota, USA 15 4Mid-Central Research and Outreach Center, University of Minnesota, Willmar, Minnesota, 16 USA 17 18 ‡Co-first authors contributed equally to the manuscript. 19 *Corresponding author: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.19.957092; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 ABSTRACT 21 Host-associated communities of bacteria (microbiota) substantially contribute to the 22 overall poultry health and performance. Gut microbiota are known to play roles in resistance to 23 pathogen infection and optimal weight gain in turkey flocks. However, knowledge of turkey 24 respiratory microbiota and its link to gut microbiota is lacking. This study presents a 16S rRNA 25 gene-based census of the turkey respiratory microbiota (nasal cavity and trachea) alongside gut 26 microbiota (cecum and ileum) in two identical commercial Hybrid Converter turkey flocks raised 27 in parallel under typical field commercial conditions. The flocks were housed in adjacent barns 28 during the brood stage and in geographically separated farms during the grow-out stage. Several 29 bacterial taxa that were acquired in the respiratory tract (RT) at the beginning of the brood stage 30 persisted throughout the flock cycle, primarily Staphylococcus. Late-emerging predominant taxa 31 in RT included Deinococcus and Corynebacterium. Tracheal and nasal microbiota of turkeys 32 were identifiably distinct from one another and from gut microbiota. Nevertheless, gut and RT 33 microbiota changed in parallel over time and appeared to share many taxa. During the brood 34 stage, the two flocks generally acquired similar gut and RT microbiota, and their average body 35 weights were comparable. Separating the flocks during the grow-out stage resulted in divergent 36 microbial profiles and body weight gain trajectories. Lower weight gain corresponded with 37 emergence of Deinococcus and Ornithobacterium in RT, and Fusobacterium and Parasutterella 38 in gut. This study provides an overview of turkey microbiota under field conditions and suggests 39 several hypotheses concerning the respiratory microbiome. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.19.957092; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 40 IMPORTANCE 41 Turkey meat is an important source of animal protein, and the industry around its production 42 contributes significantly to the agricultural economy. The nonpathogenic symbionts present in 43 the gut of turkeys are known to impact bird health and flock performance. However, the 44 respiratory microbiota in turkeys are entirely unexplored. This study has elucidated the 45 microbiota of respiratory tracts of turkeys from two commercial flocks raised in parallel 46 throughout a normal flock cycle. Further, the study suggests that bacteria originating in the gut or 47 in poultry house environments may influence respiratory communities and consequently induce 48 poor performance, either directly or indirectly. Future attempts to develop microbiome-based 49 interventions for turkey health should delimit the contributions of respiratory microbiota and aim 50 to limit disturbances to those communities. 51 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.19.957092; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 52 INTRODUCTION 53 Bacterial communities (microbiota) associated with poultry have great influences on bird 54 development (1, 2), health (3–5), and production performance (6, 7). A wealth of information on 55 microbial density and composition in the gastrointestinal tracts of broiler chickens, layer 56 chickens, and turkeys has been generated in the last decade (3, 4, 8–10). Certain bacterial species 57 in the turkey gut are associated with adverse effects such as increased susceptibility to pathogens 58 (11–13), inefficient feed conversion (1), and suboptimal market weights (6, 14), while other 59 species are correlated with improved health and enhanced performance metrics, including weight 60 gain (1, 6, 15, 16). 61 Microbiota composition in the turkey gut is dependent on a number of intrinsic and 62 extrinsic factors. Intrinsic factors such as bird age and the site within the gut (cecum, ileum, 63 duodenum, jejunum, ileum, etc.) are good predictors of the diversity and composition of 64 communities in commercial turkeys (6, 17, 18). Gradual parallel composition changes occur in 65 different gut sites as the bird ages (6, 17, 18), reflecting anatomical differences between sites and 66 age-dependent physiological changes. Possible extrinsic factors are farm location (19), flock 67 rearing and health management practices (15, 20, 21), including diet, housing conditions (22), 68 litter reuse (10, 23, 24), and vaccination and antibiotic use (25). These factors do cause 69 microbiota composition changes, but the effects of those changes on production performance 70 metrics are variable and thus difficult to predict. In particular, we have shown that the gut 71 microbial composition of multiple chicken flocks of the same breed and raised adjacently may 72 differentiate from one another with age (26), potentially leading to a performance gap between 73 flocks. Such detailed information is lacking for turkey flocks. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.19.957092; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 74 A thorough understanding of the gut and respiratory microbiota is critical to decipher the 75 potential roles of microbiota in pathogen resistance in addition to production performance. Our 76 previous work in chickens demonstrated that the microbiota of the respiratory system overlap 77 substantially with gastrointestinal microbiota (26, 27). We observed that numerous taxa reside 78 simultaneously in the respiratory and gastrointestinal tracts and shift in parallel over time (26, 79 27). It is vitally important to establish whether these dynamics are true in turkeys as well, as they 80 may indicate how abrupt changes to flock management disrupt both systems. 81 Currently, there is no exposition of turkey respiratory microbiota that would permit 82 comparison with gut microbiota. Previous studies of turkey respiratory bacteria have focused on 83 specific pathogens, such as Mycoplasma (28, 29) and Ornithobacterium rhinotracheale (30) and 84 have not considered non-pathogenic microbial communities residing therein. In other poultry, 85 different respiratory sites are known to house distinct bacterial communities that develop with 86 age (26, 27, 31, 32), but the contribution of these communities to poultry health is largely 87 unknown. Of particular interest are the microbiota of the upper respiratory system (the nasal 88 cavity and the trachea), as these sites are among the first points of contact for airborne bacteria, 89 including aerosolized fecal bacteria (33). Understanding how upper respiratory microbiota 90 develops alongside lower gastrointestinal microbiota is critical to the development of effective 91 interventions against transmission of performance-inhibiting pathogens and to improve poultry 92 health and productivity. 93 In the current study, we provide respiratory and gastrointestinal microbiota observed in 94 two identical commercial turkey flocks from a vertically integrated turkey production system. 95 The birds were raised in adjacent barns throughout the brood farm stage but were geographically 96 separated for the grow-out/finisher farm stage. This management setup allowed parallel 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.19.957092; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 97 observation of weight gain and microbiota both before and after separation and transfer to new 98 locations. 99 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.19.957092; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.