Digestive and respiratory tracts are inhabited by rich bacterial communities that can vary between their different segments. In comparison with other bird taxa with developed caeca, parrots that lack caeca have relatively lower variability in intestinal morphology. Here, based on 16S rRNA metabarcoding, we describe variation in microbiota across different parts of parrot digestive and respiratory tracts both at interspecies and intraspecies levels. In domesticated budgerigar (Melopsittacus undulatus), we describe the bacterial variation across eight selected sections of respiratory and digestive tracts, and three non-destructively collected sample types (faeces, and cloacal and oral swabs). Our results show important microbiota divergence between the upper and lower digestive tract, but similarities between respiratory tract and crop, and also between different intestinal segments. Faecal samples appear to provide a better proxy for intestinal microbiota composition than the cloacal swabs. Oral swabs had a similar bacterial composition as the crop and trachea. For a subset of tissues, we confirmed the same pattern also in six different parrot species. Finally, using the faeces and oral swabs in budgerigars, we revealed high oral, but low faecal microbiota stability during a 3-week period mimicking pre-experiment acclimation. Our findings provide a basis essential for microbiota-related experimental planning and result generalisation in non-poultry birds.

Department of Zoology Faculty of Science Charles University Prague Czech Republic

Institute of Vertebrate Biology Czech Academy of Sciences Brno Czech Republic

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Abundo MEC, Ngunjiri JM, Taylor KJM, Ji H, Ghorbani A, K. C. M, Weber BP, Johnson TJ, Lee CW. Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types. PLOS ONE. 2021;16(1):e0241732. doi: 10.1371/journal.pone.0241732. PubMed DOI PMC

Adil S, Magray SN. Impact and manipulation of gut microflora in poultry: a review. J of Animal and Veterinary Advances. 2012;11:873–877. doi: 10.3923/javaa.2012.873.877. DOI

Albenberg L, Esipova TV, Judge CP, et al. Correlation between intraluminal oxygen gradient and radial partitioning of intestinal microbiota. Gastroenterology. 2014;147:1055–1063.e8. doi: 10.1053/j.gastro.2014.07.020. PubMed DOI PMC

Alcaraz LD, Hernández AM, Peimbert M. Exploring the cockatiel (Nymphicus hollandicus) fecal microbiome, bacterial inhabitants of a worldwide pet. PeerJ. 2016;4:e2837. doi: 10.7717/peerj.2837. PubMed DOI PMC

Bäckhed F, Ley RE, Sonnenburg JL, et al. Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–1920. doi: 10.1126/science.1104816. PubMed DOI

Balsamo G, Maxted AM, Midla JW, et al. Compendium of measures to control Chlamydia psittaci infection among humans (psittacosis) and pet birds (avian chlamydiosis), 2017. J Avian Med Surg. 2017;31:262–282. doi: 10.1647/217-265. PubMed DOI

Bates D, Mächler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. Journal of Statistical Software. 2015;67:1–48. doi: 10.18637/jss.v067.i01. DOI

Bell SE, Nash AK, Zanghi BM, et al. An assessment of the stability of the canine oral microbiota after probiotic administration in healthy dogs over time. Front Vet Sci. 2020;7:616. doi: 10.3389/fvets.2020.00616. PubMed DOI PMC

Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society: Series B (Methodological) 1995;57:289–300. doi: 10.1111/j.2517-6161.1995.tb02031.x. DOI

Benskin C, Rhodes G, Pickup RW, et al. Diversity and temporal stability of bacterial communities in a model passerine bird, the zebra finch. Mol Ecol. 2010;19:5531–5544. doi: 10.1111/j.1365-294X.2010.04892.x. PubMed DOI

Berlow M, Kohl KD, Derryberry EP. Evaluation of non-lethal gut microbiome sampling methods in a passerine bird. Ibis. 2020;162:911–923. doi: 10.1111/ibi.12807. DOI

Bobbie CB, Mykytczuk NCS, Schulte-Hostedde AI. Temporal variation of the microbiome is dependent on body region in a wild mammal (Tamiasciurus hudsonicus) FEMS Microbiology Ecology. 2017;93:fix081. doi: 10.1093/femsec/fix081. PubMed DOI

Bodawatta KH, Sam K, Jønsson KA, Poulsen M. Comparative analyses of the digestive tract microbiota of New Guinean passerine birds. Front Microbiol. 2018;9:1830. doi: 10.3389/fmicb.2018.01830. PubMed DOI PMC

Bodawatta KH, Puzejova K, Sam K, et al. Cloacal swabs and alcohol bird specimens are good proxies for compositional analyses of gut microbial communities of Great tits (Parus major) anim microbiome. 2020;2:9. doi: 10.1186/s42523-020-00026-8. PubMed DOI PMC

Callahan BJ, McMurdie PJ, Rosen MJ, et al. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Meth. 2016;13:581–583. doi: 10.1038/nmeth.3869. PubMed DOI PMC

Cameron SJS, Huws SA, Hegarty MJ, et al. The human salivary microbiome exhibits temporal stability in bacterial diversity. FEMS Microbiology Ecology. 2015;91:9. doi: 10.1093/femsec/fiv091. PubMed DOI

Das B, Nair GB. Homeostasis and dysbiosis of the gut microbiome in health and disease. J Biosci. 2019;44:117. doi: 10.1007/s12038-019-9926-y. PubMed DOI

Davis NM, Proctor DM, Holmes SP, et al. Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome. 2018;6:1–14. doi: 10.1186/s40168-018-0605-2. PubMed DOI PMC

Divín D, Goméz Samblas M, Kuttiyarthu Veetil N, et al. Cannabinoid receptor 2 evolutionary gene loss makes parrots more susceptible to neuroinflammation. Proceedings of the Royal Society B: Biological Sciences. 2022;289:20221941. doi: 10.1098/rspb.2022.1941. PubMed DOI PMC

Drovetski SV, O’Mahoney M, Ransome EJ, et al. Spatial organization of the gastrointestinal microbiota in urban Canada geese. Sci Rep. 2018;8:3713. doi: 10.1038/s41598-018-21892-y. PubMed DOI PMC

Drovetski SV, O’Mahoney MJV, Matterson KO, et al. Distinct microbiotas of anatomical gut regions display idiosyncratic seasonal variation in an avian folivore. Animal Microbiome. 2019;1:2. doi: 10.1186/s42523-019-0002-6. PubMed DOI PMC

Edgar RC, Haas BJ, Clemente JC, et al. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011;27:2194–2200. doi: 10.1093/bioinformatics/btr381. PubMed DOI PMC

Garcia-Mazcorro JF, Castillo-Carranza SA, Guard B, et al. Comprehensive molecular characterization of bacterial communities in feces of pet birds using 16S marker sequencing. Microb Ecol. 2017;73:224–235. doi: 10.1007/s00248-016-0840-7. PubMed DOI

Garcia-Mazcorro JF, Alanis-Lopez C, Marroquin-Cardona AG, Kawas JR. Composition and potential function of fecal bacterial microbiota from six bird species. Birds. 2021;2:42–59. doi: 10.3390/birds2010003. DOI

Glendinning L, McLachlan G, Vervelde L. Age-related differences in the respiratory microbiota of chickens. PLOS ONE. 2017;12:e0188455. doi: 10.1371/journal.pone.0188455. PubMed DOI PMC

Grond K, Guilani H, Hird SM. Spatial heterogeneity of the shorebird gastrointestinal microbiome. Royal Society Open Science. 2020;7:191609. doi: 10.1098/rsos.191609. PubMed DOI PMC

Hird SM, Sánchez C, Carstens BC, Brumfield RT. Comparative gut microbiota of 59 neotropical bird species. Front Microbiol. 2015;2015:1403. doi: 10.3389/fmicb.2015.01403. PubMed DOI PMC

Hu W, Pasare C. Location, location, location: tissue-specific regulation of immune responses. Journal of Leukocyte Biology. 2013;94:409–421. doi: 10.1189/jlb.0413207. PubMed DOI PMC

Jacobs L, McMahon BH, Berendzen J, et al. California condor microbiomes: bacterial variety and functional properties in captive-bred individuals. PLOS ONE. 2019;14:e0225858. doi: 10.1371/journal.pone.0225858. PubMed DOI PMC

Jiang H, Lei R, Ding S-W, Zhu S. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics. 2014;15:182. doi: 10.1186/1471-2105-15-182. PubMed DOI PMC

Jones JC, Sonnberg S, Koçer ZA, et al. Possible role of songbirds and parakeets in transmission of influenza A (H7N9) virus to humans. Emerg Infect Dis. 2014;20:380–385. doi: 10.3201/eid2003.131271. PubMed DOI PMC

Klindworth A, Pruesse E, Schweer T, et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013;41:e1. doi: 10.1093/nar/gks808. PubMed DOI PMC

Koch H, Schmid-Hempel P. Socially transmitted gut microbiota protect bumble bees against an intestinal parasite. Proc Natl Acad Sci U S A. 2011;108:19288–19292. doi: 10.1073/pnas.1110474108. PubMed DOI PMC

Kreisinger J, Kropáčková L, Petrželková A, et al. Temporal stability and the effect of transgenerational transfer on fecal microbiota structure in a long distance migratory bird. Front Microbiol. 2017;8:50. doi: 10.3389/fmicb.2017.00050. PubMed DOI PMC

Kropáčková L, Pechmanová H, Vinkler M, et al. Variation between the oral and faecal microbiota in a free-living passerine bird, the great tit (Parus major) PLOS ONE. 2017;12:e0179945. doi: 10.1371/journal.pone.0179945. PubMed DOI PMC

Kropáčková L, Těšický M, Albrecht T, et al. Co-diversification of gastrointestinal microbiota and phylogeny in passerines is not explained by ecological divergence. Mol Ecol. 2017;26:5292–5304. doi: 10.1111/mec.14144. PubMed DOI

Kursa O, Tomczyk G, Sawicka-Durkalec A, et al. Bacterial communities of the upper respiratory tract of turkeys. Sci Rep. 2021;11:2544. doi: 10.1038/s41598-021-81984-0. PubMed DOI PMC

Laviad-Shitrit S, Izhaki I, Lalzar M, Halpern M. Comparative analysis of intestine microbiota of four wild waterbird species. Front Microbiol. 2019;10:1911. doi: 10.3389/fmicb.2019.01911. PubMed DOI PMC

Ley RE, Hamady M, Lozupone C, et al. Evolution of mammals and their gut microbes. Science. 2008;320:1647–1651. doi: 10.1126/science.1155725. PubMed DOI PMC

Liu H, Chen Z, Gao G, et al. Characterization and comparison of gut microbiomes in nine species of parrots in captivity. Symbiosis. 2019;78:241–250. doi: 10.1007/s13199-019-00613-7. DOI

Man WH, de Steenhuijsen Piters WAA, Bogaert D. The microbiota of the respiratory tract: gatekeeper to respiratory health. Nat Rev Microbiol. 2017;15:259–270. doi: 10.1038/nrmicro.2017.14. PubMed DOI PMC

Matějková T, Hájková P, Stopková R, et al. Oral and vaginal microbiota in selected field mice of the genus Apodemus : a wild population study. Sci Rep. 2020;10:13246. doi: 10.1038/s41598-020-70249-x. PubMed DOI PMC

McArtor DB, Lubke GH, Bergeman CS. Extending multivariate distance matrix regression with an effect size measure and the asymptotic null distribution of the test statistic. Psychometrika. 2017;82:1052–1077. doi: 10.1007/s11336-016-9527-8. PubMed DOI PMC

Mulholland KA, Robinson MG, Keeler SJ, et al. Metagenomic analysis of the respiratory microbiome of a broiler flock from hatching to processing. Microorganisms. 2021;9:721. doi: 10.3390/microorganisms9040721. PubMed DOI PMC

Nakagawa S, Schielzeth H. A general and simple method for obtaining R2 from generalized linear mixed-effects models. Methods Ecol Evol. 2013;4:133–142. doi: 10.1111/j.2041-210x.2012.00261.x. DOI

Nga VT, Ngoc TU, Minh LB, et al. Zoonotic diseases from birds to humans in Vietnam: possible diseases and their associated risk factors. Eur J Clin Microbiol Infect Dis. 2019;38:1047–1058. doi: 10.1007/s10096-019-03505-2. PubMed DOI

Ngunjiri JM, Taylor KJM, Abundo MC, et al. Farm stage, bird age, and body site dominantly affect the quantity, taxonomic composition, and dynamics of respiratory and gut microbiota of commercial layer chickens. Appl Environ Microbiol. 2019;85:e03137. doi: 10.1128/AEM.03137-18. PubMed DOI PMC

Ost KS, Round JL. Communication between the microbiota and mammalian immunity. Annu Rev Microbiol. 2018;72:399–422. doi: 10.1146/annurev-micro-090817-062307. PubMed DOI PMC

Pascoe EL, Hauffe HC, Marchesi JR, Perkins SE. Network analysis of gut microbiota literature: an overview of the research landscape in non-human animal studies. ISME J. 2017;11:2644–2651. doi: 10.1038/ismej.2017.133. PubMed DOI PMC

Perry EK, Digby A, Taylor MW. The low-diversity fecal microbiota of the critically endangered kākāpō is robust to anthropogenic dietary and geographic influences. Front Microbiol. 2017;8:2033. doi: 10.3389/fmicb.2017.02033. PubMed DOI PMC

Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590–D596. doi: 10.1093/nar/gks1219. PubMed DOI PMC

R Core Team . R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2020.

Ravichandran K, Anbazhagan S, Karthik K, et al. A comprehensive review on avian chlamydiosis: a neglected zoonotic disease. Trop Anim Health Prod. 2021;53:414. doi: 10.1007/s11250-021-02859-0. PubMed DOI PMC

Reikvam DH, Erofeev A, Sandvik A, et al. Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression. PLoS ONE. 2011;6:e17996. doi: 10.1371/journal.pone.0017996. PubMed DOI PMC

Schmiedová L, Kreisinger J, Požgayová M, et al. Gut microbiota in a host–brood parasite system: insights from common cuckoos raised by two warbler species. FEMS Microbiol Ecol. 2020;96:fiaa143. doi: 10.1093/femsec/fiaa143. PubMed DOI

Song SJ, Sanders JG, Delsuc F, et al. Comparative analyses of vertebrate gut microbiomes reveal convergence between birds and bats. mBio. 2020;11:e02901. doi: 10.1128/mBio.02901-19. PubMed DOI PMC

Sottas C, Schmiedová L, Kreisinger J, et al. Gut microbiota in two recently diverged passerine species: evaluating the effects of species identity, habitat use and geographic distance. BMC Ecology and Evolution. 2021;21:41. doi: 10.1186/s12862-021-01773-1. PubMed DOI PMC

Sterneberg-van der Maaten T, Turner D, Van Tilburg J, Vaarten J. Benefits and risks for people and livestock of keeping companion animals: searching for a healthy balance. Journal of Comparative Pathology. 2016;155:S8–S17. doi: 10.1016/j.jcpa.2015.06.007. PubMed DOI

Strandwitz P. Neurotransmitter modulation by the gut microbiota. Brain Res. 2018;1693:128–133. doi: 10.1016/j.brainres.2018.03.015. PubMed DOI PMC

Taylor MJ, Mannan RW, U’Ren JM, et al. Age-related variation in the oral microbiome of urban Cooper’s hawks (Accipiter cooperii) BMC Microbiol. 2019;19:47. doi: 10.1186/s12866-019-1413-y. PubMed DOI PMC

Taylor KJM, Ngunjiri JM, Abundo MC, et al. Respiratory and gut microbiota in commercial turkey flocks with disparate weight gain trajectories display differential compositional dynamics. Applied and Environmental Microbiology. 2020;86:e00431–e00520. doi: 10.1128/AEM.00431-20. PubMed DOI PMC

Videvall E, Strandh M, Engelbrecht A, et al. Measuring the gut microbiome in birds: comparison of faecal and cloacal sampling. Molecular Ecology Resources. 2018;18:424–434. doi: 10.1111/1755-0998.12744. PubMed DOI

Vogtmann E, Hua X, Zhou L, et al. Temporal variability of oral microbiota over 10 months and the implications for future epidemiologic studies. Cancer Epidemiol Biomarkers Prev. 2018;27:594–600. doi: 10.1158/1055-9965.EPI-17-1004. PubMed DOI PMC

Waite DW, Deines P, Taylor MW. Gut microbiome of the critically endangered New Zealand parrot, the kakapo (Strigops habroptilus) PLoS ONE. 2012;7:e35803. doi: 10.1371/journal.pone.0035803. PubMed DOI PMC

Waite DW, Dsouza M, Sekiguchi Y, et al. Network-guided genomic and metagenomic analysis of the faecal microbiota of the critically endangered kakapo. Sci Rep. 2018;8:8128. doi: 10.1038/s41598-018-26484-4. PubMed DOI PMC

Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007;73:5261–5267. doi: 10.1128/AEM.00062-07. PubMed DOI PMC

Wilkinson N, Hughes RJ, Aspden WJ, et al. The gastrointestinal tract microbiota of the Japanese quail, Coturnix japonica. Appl Microbiol Biotechnol. 2016;100:4201–4209. doi: 10.1007/s00253-015-7280-z. PubMed DOI

Williams T, Athrey G. Cloacal swabs are unreliable sources for estimating lower gastro-intestinal tract microbiota membership and structure in broiler chickens. Microorganisms. 2020;8:718. doi: 10.3390/microorganisms8050718. PubMed DOI PMC

Xenoulis PG, Gray PL, Brightsmith D, et al. Molecular characterization of the cloacal microbiota of wild and captive parrots. Vet Microbiol. 2010;146:320–325. doi: 10.1016/j.vetmic.2010.05.024. PubMed DOI

Are there consistent effects of gut microbiota composition on performance, productivity and condition in poultry?