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Respiratory Bacterial Microbiota in Cattle

From Development to Modulation to Enhance Respiratory Health
  • Edouard Timsit
    Correspondence
    Corresponding author.
    Affiliations
    Ceva Santé Animale, 10 Avenue de la Ballastière, Libourne 33500, France
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  • Author Footnotes
    1 Present address: 3280 Hospital Drive Northwest, Calgary, Alberta T2N 4Z6, Canada.
    Chris McMullen
    Footnotes
    1 Present address: 3280 Hospital Drive Northwest, Calgary, Alberta T2N 4Z6, Canada.
    Affiliations
    Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada
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  • Author Footnotes
    2 Present address: Room 3-60D1 Ag/For Center Edmonton, Alberta T6G 2P5, Canada.
    Samat Amat
    Footnotes
    2 Present address: Room 3-60D1 Ag/For Center Edmonton, Alberta T6G 2P5, Canada.
    Affiliations
    Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada

    Lethbridge Research and Development Center, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada

    Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada
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  • Author Footnotes
    3 Present address: 5403 1st Avenue South, Lethbridge, Alberta T1J 4B1, Canada.
    Trevor W. Alexander
    Footnotes
    3 Present address: 5403 1st Avenue South, Lethbridge, Alberta T1J 4B1, Canada.
    Affiliations
    Lethbridge Research and Development Center, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada
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  • Author Footnotes
    1 Present address: 3280 Hospital Drive Northwest, Calgary, Alberta T2N 4Z6, Canada.
    2 Present address: Room 3-60D1 Ag/For Center Edmonton, Alberta T6G 2P5, Canada.
    3 Present address: 5403 1st Avenue South, Lethbridge, Alberta T1J 4B1, Canada.

      Keywords

      Key points

      • The respiratory bacterial microbiota is dynamic, changing significantly during periods of increased risk for bovine respiratory disease.
      • The respiratory microbiota is inhabited predominantly by 5 bacterial phyla: Proteobacteria, Firmicutes, Tenericutes, Actinobacteria, and Bacteroidetes; the relative abundance of each differs by animal age and production system.
      • Upper respiratory tract and lower respiratory tract microbiotas differ in diversity and composition. The nasopharyngeal microbiota contributes the most to the lower respiratory microbiota and thus should be the primary target for sampling or modulation strategies.
      • Composition of the respiratory microbiota is associated with respiratory health; increased abundances of respiratory Lactobacillus and/or Lactococcus are associated with good respiratory health.
      • Intranasal application of selected Lactobacillus strains modifies the composition of the nasopharyngeal microbiotas in cattle and can provide colonization resistance against opportunistic bacterial pathogens such as Mannheimia haemolytica.

      Introduction

      Over the past decade it has become clear that mammals live in symbiosis with their abundant resident microbes.
      • Turnbaugh P.J.
      • Ley R.E.
      • Hamady M.
      • et al.
      The human microbiome project.
      Advances in culture-independent techniques (eg, 16S ribosomal RNA [rRNA] sequencing) have enabled detection and quantification of bacterial species that are difficult or impossible to detect by culture-based methods (Box 1).
      • Holman D.B.
      • Timsit E.
      • Alexander T.W.
      The nasopharyngeal microbiota of feedlot cattle.
      These advances in the field of molecular techniques, in particular metagenomics, have led to the definition of the animal microbiota, a term that refers to the complex microbial ecosystems in and on bodies of animals.
      • Turnbaugh P.J.
      • Ley R.E.
      • Hamady M.
      • et al.
      The human microbiome project.
      16S rRNA sequencing
      Typically, short segments of the 16S rRNA that include hypervariable regions are sequenced for bacterial classification in microbiota studies (Fig. 1). Therefore, composition of the respiratory microbiota often is reported at the phylum, family, or genus level but not at the species level because only a small proportion (30%–50%) of these short 16S rRNA sequences can be classified as OTUs beyond the genus level.
      Figure thumbnail gr1
      Fig. 1Schematic illustration of basic workflow for respiratory microbiota research (16S rRNA sequencing). Only a small proportion of the OTUs are classified at the species level (30%–50%). BAL, bronchoalveolar lavage; DNS, deep nasal swab; TTA, transtracheal aspiration.
      (Adapted from Jo JH, Kennedy EA, Kong H. Research techniques made simple: bacterial 16s ribosomal RNA gene sequencing in cutaneous research. J Invest Dermatol. 2016; 136, e23ee27; with permission).
      Like other body sites, the respiratory tract of cattle is colonized by a variety of different bacterial microbiotas directly after birth.
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      Composition and diversity of these microbiotas have been recently associated with respiratory health in cattle.
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      ,
      • Timsit E.
      • Holman D.B.
      • Hallewell J.
      • et al.
      The nasopharyngeal microbiota in feedlot cattle and its role in respiratory health.
      More specifically, airway microbiotas enriched with known beneficial bacteria, such as Lactobacillus, have been associated with good respiratory health,
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      ,
      • Holman D.B.
      • McAllister T.A.
      • Topp E.
      • et al.
      The nasopharyngeal microbiota of feedlot cattle that develop bovine respiratory disease.
      whereas microbiotas enriched with known bacterial pathogens, such as Mycoplasma bovis, Mannheimia haemolytica, or Pasteurella multocida, have been associated with bovine respiratory disease (BRD).
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      ,
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      Investigating the role of the respiratory microbiota in health and disease is a relatively new, rapidly developing field of research that provides new opportunities for the prevention and treatment of BRD.
      • Koppen I.J.N.
      • Bosch A.
      • Sanders E.A.M.
      • et al.
      The respiratory microbiota during health and disease: a paediatric perspective.
      This review summarizes current knowledge regarding composition of the respiratory bacterial microbiota in dairy cattle and beef cattle and its relationship with the development of BRD. Approaches to modulate the respiratory bacterial microbiota to promote enhanced heath (eg, probiotics, bacteriophages, and prebiotics) also are discussed.

      Composition of the bacterial respiratory microbiota in healthy cattle

      The diversity of bacteria on earth is vast, comprising 55 phyla.
      • Ley R.E.
      • Peterson D.A.
      • Gordon J.I.
      Ecological and evolutionary forces shaping microbial diversity in the human intestine.
      The cattle respiratory tract is inhabited predominantly by 5 of these phyla (Proteobacteria, Firmicutes, Tenericutes, Actinobacteria, and Bacteroidetes [Table 1]), which underlines its suitability for the growth of only a limited number of bacteria. This diversity is largely due to the biophysical properties of respiratory mucosal surfaces, that is, temperature, moisture, and pH.
      • Wypych T.P.
      • Wickramasinghe L.C.
      • Marsland B.J.
      The influence of the microbiome on respiratory health.
      Table 1Composition of the nasopharyngeal microbiota in preweaned and postweaned beef calves and preweaned dairy calves
      Data from Refs.
      • Holman D.B.
      • Timsit E.
      • Alexander T.W.
      The nasopharyngeal microbiota of feedlot cattle.
      ,
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      ,
      • Hall J.A.
      • Isaiah A.
      • Estill C.T.
      • et al.
      Weaned beef calves fed selenium-biofortified alfalfa hay have an enriched nasal microbiota compared with healthy controls.
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      • Timsit E.
      • Workentine M.
      • Schryvers A.B.
      • et al.
      Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot.
      ,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      ,
      • Amat S.
      • Holman D.B.
      • Timsit E.
      • et al.
      Evaluation of the nasopharyngeal microbiota in beef cattle transported to a feedlot, with a focus on lactic acid-producing bacteria.
      ,
      • Timsit E.
      • Workentine M.
      • Crepieux T.
      • et al.
      Effects of nasal instillation of a nitric oxide-releasing solution or parenteral administration of tilmicosin on the nasopharyngeal microbiota of beef feedlot cattle at high-risk of developing respiratory tract disease.
      ,
      • Zeineldin M.
      • Lowe J.
      • de Godoy M.
      • et al.
      Disparity in the nasopharyngeal microbiota between healthy cattle on feed, at entry processing and with respiratory disease.
      ,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Comparison of the nasopharyngeal bacterial microbiota of beef calves raised without the use of antimicrobials between healthy calves and those diagnosed with bovine respiratory disease.
      TypeStudy DesignPhylum LevelGenus LevelReference
      Only studies reporting the general composition of the nasopharyngeal microbiota are presented.
      Postweaned feedlot beef calvesCross-sectional; 2 populations (BRD, n = 82; healthy, n = 82)Proteobacteria (69.3%), Tenericutes (22.5%), Firmicutes (3.3%), Actinobacteria (2.3%), Bacteroidetes (2.3%)Mycoplasma (22.2%), Moraxella (19.5%), Histophilus (19.0%), Psychrobacter (9.8%), Mannheimia (6.3%), Pasteurella (4.4%), Pseudomonas (1.8%)McMullen et al,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Comparison of the nasopharyngeal bacterial microbiota of beef calves raised without the use of antimicrobials between healthy calves and those diagnosed with bovine respiratory disease.
      2019
      Longitudinal; 4 populations (BRD at entry, n = 22; BRD at diagnosis; n = 22, healthy at entry, n = 44; healthy at diagnosis, n = 10)Proteobacteria (34.8%), Firmicutes (18.6%), Actinobacteria (17.2%), Bacteroidetes (12.1%), Tenericutes (11.2%), Fusobacteria (1.2%)Moraxella (10.9%), Mycoplasma (10.7%), Acinetobacter (9.7%), Rathayibacter (5.0%), Promicromonospora (4.4%), Mannheimia (4.1%), Solibacillus (3.5%), Clostridium (3.3%), Corynebacterium (3.8%), Pasteurella (1.9%)Zeineldin et al,
      • Zeineldin M.
      • Lowe J.
      • de Godoy M.
      • et al.
      Disparity in the nasopharyngeal microbiota between healthy cattle on feed, at entry processing and with respiratory disease.
      2017
      Longitudinal; 2 populations of 30 calves sampled at the ranch (d 0), at feedlot entry (d 2), and on d 7 and d 28 after entryTenericutes (41.1%), Proteobacteria (31.8%), Firmicutes (4.6%)Mycoplasma (40.8%), Moraxella (18.7%), Pasteurella (6.8%), Mannheimia (3.8%)Stroebel et al,
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      2018
      Longitudinal; 1 population of 4 calves sampled at feedlot entry and on d 60 after entryProteobacteria (68.9%), Firmicutes (19.2%)At entry: Pseudomonas (23.7%), Shewanella (23.5%), Acinetobacter (17.5%), Carnobacterium (12.2%). At d 60: Staphylococcus (20.8%), Mycoplasma (14.9%), Mannheimia (10.4%), Moraxella (9.4%)Holman et al,
      • Holman D.B.
      • Timsit E.
      • Alexander T.W.
      The nasopharyngeal microbiota of feedlot cattle.
      2015
      Longitudinal; 1 population of 30 calves sampled at the ranch (d 0), at feedlot entry (d 2), and on d 40 after entryTenericutes (53.2%), Proteobacteria (34.7%), Firmicutes (4.2%), Bacteroidetes (3.7%), Actinobacteria (3.4%)NRTimsit et al,
      • Timsit E.
      • Workentine M.
      • Schryvers A.B.
      • et al.
      Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot.
      2016
      Longitudinal; 2 populations (treated with NORS [n = 10] or tilmicosin [n = 10] at entry) sampled at entry, and on d 1, d 5, and d 10 after entryTenericutes (92.8%), Proteobacteria (5.9%), Firmicutes (0.6%), Actinobacteria (0.6%), Bacteroidetes (0.1%)NRTimsit et al,
      • Timsit E.
      • Workentine M.
      • Crepieux T.
      • et al.
      Effects of nasal instillation of a nitric oxide-releasing solution or parenteral administration of tilmicosin on the nasopharyngeal microbiota of beef feedlot cattle at high-risk of developing respiratory tract disease.
      2017
      Cross-sectional; 3 populations (controls, n = 5; medium selenium, n = 6; high selenium, n = 5)Proteobacteria (31.7%), Bacteroidetes (27.5%), Firmicutes (24.3%), Actinobacteria (7.1%), Tenericutes (4.4%)NRHall et al,
      • Hall J.A.
      • Isaiah A.
      • Estill C.T.
      • et al.
      Weaned beef calves fed selenium-biofortified alfalfa hay have an enriched nasal microbiota compared with healthy controls.
      2017
      Longitudinal; 1 population of 13 calves sampled at the ranch (d 0), at feedlot entry (d 2) and on d 5 and d 12 after entryProteobacteria (36.1%), Firmicutes (20.1%), Tenericutes (19.3%), Actinobacteria (12.7%), Bacteroidetes (8.6%)NRAmat et al,
      • Amat S.
      • Holman D.B.
      • Timsit E.
      • et al.
      Evaluation of the nasopharyngeal microbiota in beef cattle transported to a feedlot, with a focus on lactic acid-producing bacteria.
      2019
      Preweaned and postweaned beef calvesLongitudinal; 3 populations of 40 calves sampled at initial vaccination, weaning and on d 40 after entry at feedlotsProteobacteria (27.5%), Actinobacteria (25.9%), Tenericutes (24.3%), Firmicutes (13.5%)Mycoplasma (24.1%), Lactococcus (10.7%), Moraxella (7.4%), Histophilus (6.78%), Pasteurella (6.0%)McMullen et al,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      2018
      Preweaned dairy calvesLongitudinal; 1 population of 81 calves sampled at 3 d, 14 d, and 35 d of ageProteobacteria (52.1%), Firmicutes (23.0%), Bacteroidetes (11.4%), Tenericutes (3.4%)NRLima et al,
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      2019
      Abbreviation: NR, data not reported; NORS, nitric oxide releasing solution.
      a Only studies reporting the general composition of the nasopharyngeal microbiota are presented.
      The composition of the airway microbiotas evolves over time due to a variety of selection pressures, which further influence the colonization process of the respiratory tract, including (Fig. 2) (1) endogenous forces, such as mucus, IgA, and innate/adaptative immune recognition,
      • Wypych T.P.
      • Wickramasinghe L.C.
      • Marsland B.J.
      The influence of the microbiome on respiratory health.
      and (2) exogenous forces, such as the maternal vaginal microbiota,
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      environmental biodiversity,
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      diet,
      • Hall J.A.
      • Isaiah A.
      • Estill C.T.
      • et al.
      Weaned beef calves fed selenium-biofortified alfalfa hay have an enriched nasal microbiota compared with healthy controls.
      infection,
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      ,
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      stressful events (weaning, transportation, and commingling)
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      • Timsit E.
      • Workentine M.
      • Schryvers A.B.
      • et al.
      Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot.
      • Holman D.B.
      • Timsit E.
      • Amat S.
      • et al.
      The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.
      and parenteral antibiotics.
      • Holman D.B.
      • Yang W.
      • Alexander T.W.
      Antibiotic treatment in feedlot cattle: a longitudinal study of the effect of oxytetracycline and tulathromycin on the fecal and nasopharyngeal microbiota.
      ,
      • Holman D.B.
      • Timsit E.
      • Booker C.W.
      • et al.
      Injectable antimicrobials in commercial feedlot cattle and their effect on the nasopharyngeal microbiota and antimicrobial resistance.
      Unfortunately, to date, no study has described the composition of the developing airway microbiotas across the life span of either dairy cattle or beef cattle.
      Figure thumbnail gr2
      Fig. 2Factors influencing the composition of the respiratory microbiota in cattle. The microbiota can develop toward a balanced, stable microbiota, which is more resistant against pathogens colonization and/or proliferation. Conversely, the microbiota also can develop toward a community that is imbalanced, less stable, and more prone to infection.
      (Adapted from van den Broek MFL, De Boeck I, Kiekens F, Boudewyns A, Vanderveken OM, Lebeer S. Translating recent microbiome insights in otitis media into probiotic strategies. Clin Microbiol Rev. 2019; 3, 32; with permission).
      A systematic review of the literature (performed in PubMed on December 12, 2019; key words [respiratory] AND [cattle] AND [microbiota OR microbiome]) revealed that most published studies have focused on the respiratory microbiota of postweaned beef cattle (n = 16), with only a limited number of studies describing the composition of the nasopharyngeal microbiota in preweaned dairy calves (n = 3)
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      ,
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      ,
      • Gaeta N.C.
      • Lima S.F.
      • Teixeira A.G.
      • et al.
      Deciphering upper respiratory tract microbiota complexity in healthy calves and calves that develop respiratory disease using shotgun metagenomics.
      or beef calves (n = 2).
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      ,
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Microbiome of the upper nasal cavity of beef calves prior to weaning.
      Furthermore, these studies focused on the upper respiratory tract (URT), with only 6 studies reporting the composition of the lower respiratory tract (LRT) microbiota (sampled by transtracheal aspiration
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      ,
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      ,
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      ,
      • Amat S.
      • Alexander T.W.
      • Holman D.B.
      • et al.
      Intranasal bacterial therapeutics reduce colonization by the respiratory pathogen Mannheimia haemolytica in dairy calves.
      or bronchoalveolar lavage [McMullen C, Alexander TW, Leguillette R, et al. Topography of the respiratory tract bacterial microbiota in feedlot beef calves, submitted for publication]
      • Zeineldin M.M.
      • Lowe J.F.
      • Grimmer E.D.
      • et al.
      Relationship between nasopharyngeal and bronchoalveolar microbial communities in clinically healthy feedlot cattle.
      ).

      The Nasopharyngeal Microbiota from Birth to 1 Month of Age in Dairy Calves

      Colonization of the airways in dairy calves begins immediately after birth and evolves quickly during the first weeks of life.
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      Abundance of bacteria in the nasopharynx (measured by the number of 16S rRNA gene copies) increases significantly from birth to 14 days of age and then either decreases slightly until day 35
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      or remains the same until day 42.
      • Osman R.
      • Malmuthuge N.
      • Gonzalez-Cano P.
      • et al.
      Development and function of the mucosal immune system in the upper respiratory tract of neonatal calves.
      The interval from birth to day 14, therefore, is highly critical for microbial establishment
      • Osman R.
      • Malmuthuge N.
      • Gonzalez-Cano P.
      • et al.
      Development and function of the mucosal immune system in the upper respiratory tract of neonatal calves.
      and can predispose calves to a healthy state or pneumonia/otitis during the first weeks of age (discussed later).
      The nasopharyngeal microbiota of preweaned dairy calves is dominated by Proteobacteria, especially at 3 days and 14 days of age, when this phylum can represent up to 70% of total bacterial diversity
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      ,
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      ,
      • Gaeta N.C.
      • Lima S.F.
      • Teixeira A.G.
      • et al.
      Deciphering upper respiratory tract microbiota complexity in healthy calves and calves that develop respiratory disease using shotgun metagenomics.
      (see Table 1). After day 14, however, the diversity (combined richness and evenness) of the nasopharynx increases, with other phyla becoming more abundant (including Tenericutes, Firmicutes, Actinobacteria, and Bacteroidetes).
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      ,
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      ,
      • Gaeta N.C.
      • Lima S.F.
      • Teixeira A.G.
      • et al.
      Deciphering upper respiratory tract microbiota complexity in healthy calves and calves that develop respiratory disease using shotgun metagenomics.
      The most abundant bacterial genera in the nasopharynx of dairy calves are Mannheimia, Moraxella, Mycoplasma, Psychrobacter, and Pseudomonas. Relative abundances of these genera change over time, with the relative abundance of Moraxella decreasing between day 14 and day 35 and the relative abundances of Mannheimia and Mycoplasma concurrently increasing substantially.
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      ,
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      In dairy calves, composition of the nasopharyngeal microbiota is highly influenced by the maternal vaginal microbiota.
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      In 81 dairy cow-calf pairs, 73%, 76%, and 87% of the bacteria detected by next-generation sequencing (ie, operational taxonomic units [OTUs]) were shared between the maternal vaginal microbiota and the calf nasopharyngeal microbiota at 3 days, 14 days, and 35 days of age, respectively.
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      The most abundant shared bacterial genera in the dam vaginal and calf nasopharyngeal samples across all sampling days were Mannheimia, Moraxella, Bacteroides, Streptococcus, and Pseudomonas.
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      The significant overlap between the 2 microbiotas was attributed to the transfer of maternal microbes to the neonate at birth via the vaginal canal. Mannheimia was found to be relatively more abundant in the vaginal microbiota of dams whose calves did not develop pneumonia and/or otitis compared with the microbiota of dams whose calves did develop disease.
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      Therefore, it appears that the prepartum higher abundance of Mannheimia in the vagina of dairy cows may confer a protective effect on the health of the respiratory tract and middle ear of their progeny.

      The Nasopharyngeal Microbiota from Initial Vaccination to Preconditioning or Weaning in Beef Cattle (ie, Preweaned Beef Cattle)

      The nasopharyngeal microbiota changes significantly between initial vaccination (approximately 40 days of age) and preconditioning (approximately 130 days of age) or weaning (approximately 150 days of age) in beef calves.
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      ,
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Microbiome of the upper nasal cavity of beef calves prior to weaning.
      At initial vaccination, the diversity of the nasopharyngeal microbiota is low, with a high abundance of bacteria from the phylum Actinobacteria (more specifically, from the Promicromonosporaceae and Microbacteriaceae families).
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      ,
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Microbiome of the upper nasal cavity of beef calves prior to weaning.
      Nasopharyngeal diversity then increases, with higher proportions of Tenericutes, Proteobacteria, and Firmicutes at the time of preconditioning or weaning.
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      ,
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Microbiome of the upper nasal cavity of beef calves prior to weaning.
      At the genera level, relative abundances of Mycoplasma,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      ,
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Microbiome of the upper nasal cavity of beef calves prior to weaning.
      , Moraxella,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      ,
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Microbiome of the upper nasal cavity of beef calves prior to weaning.
      and Psychrobacter
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Microbiome of the upper nasal cavity of beef calves prior to weaning.
      were higher at weaning than at initial vaccination. Although some commonalities of evolution among calves exist, groups of preweaned calves that were raised on different farms evolved differently (even when managed similarly),
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      ,
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Microbiome of the upper nasal cavity of beef calves prior to weaning.
      implying that factors other than age (eg, environment and contact with older animals) have important roles in development of the nasopharyngeal microbiota.

      The Nasopharyngeal Microbiota from Weaning to the First Weeks on Feed in Beef Cattle (ie, Postweaned Beef Cattle)

      The structure of the nasopharyngeal microbiota evolves significantly from weaning at the ranch to 40 days to 60 days after entrance to a feedlot.
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      • Timsit E.
      • Workentine M.
      • Schryvers A.B.
      • et al.
      Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot.
      • Holman D.B.
      • Timsit E.
      • Amat S.
      • et al.
      The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.
      • Holman D.B.
      • Yang W.
      • Alexander T.W.
      Antibiotic treatment in feedlot cattle: a longitudinal study of the effect of oxytetracycline and tulathromycin on the fecal and nasopharyngeal microbiota.
      The largest shift occurs between departure from the ranch of origin and the first 7 days on feed, with a sharp increase in diversity of the nasopharyngeal microbiota during this short interval.
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      • Timsit E.
      • Workentine M.
      • Schryvers A.B.
      • et al.
      Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot.
      • Holman D.B.
      • Timsit E.
      • Amat S.
      • et al.
      The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.
      • Holman D.B.
      • Yang W.
      • Alexander T.W.
      Antibiotic treatment in feedlot cattle: a longitudinal study of the effect of oxytetracycline and tulathromycin on the fecal and nasopharyngeal microbiota.
      For example, the number of bacterial taxons (ie, OTUs) almost doubled (100 OTUs vs 200 OTUs) in the nasopharynx of 13 beef steer calves during the 48-hour interval from leaving ranch of origin to on-arrival processing at a feedlot.
      • Amat S.
      • Holman D.B.
      • Timsit E.
      • et al.
      Evaluation of the nasopharyngeal microbiota in beef cattle transported to a feedlot, with a focus on lactic acid-producing bacteria.
      The nasopharyngeal microbiota stabilizes after the first week on feed, with diversity either remaining the same or slightly decreasing until 40 days to 60 days on feed.
      • Timsit E.
      • Workentine M.
      • Schryvers A.B.
      • et al.
      Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot.
      ,
      • Holman D.B.
      • Timsit E.
      • Amat S.
      • et al.
      The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.
      ,
      • Holman D.B.
      • Timsit E.
      • Booker C.W.
      • et al.
      Injectable antimicrobials in commercial feedlot cattle and their effect on the nasopharyngeal microbiota and antimicrobial resistance.
      The nasopharyngeal microbiota also becomes more homogeneous among cattle during this interval.
      • Holman D.B.
      • Timsit E.
      • Amat S.
      • et al.
      The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.
      ,
      • Timsit E.
      • Workentine M.
      • Crepieux T.
      • et al.
      Effects of nasal instillation of a nitric oxide-releasing solution or parenteral administration of tilmicosin on the nasopharyngeal microbiota of beef feedlot cattle at high-risk of developing respiratory tract disease.
      For example, at day 0 prior to transport, 76 OTUs were shared in the nasopharyngeal microbiota of 14 Angus-Herford cross heifers, whereas at 5 days and 12 days after arrival at a research feedlot, there were 373 and 274 OTUs, respectively, that were shared among these cattle.
      • Holman D.B.
      • Timsit E.
      • Amat S.
      • et al.
      The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.
      Numerous factors can explain significant shifts in the structure of the nasopharyngeal microbiota around cattle marketing (Fig. 2). First, transportation and adaptation to a feedlot environment have a major effect on the nasopharyngeal bacterial community.
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      • Timsit E.
      • Workentine M.
      • Schryvers A.B.
      • et al.
      Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot.
      • Holman D.B.
      • Timsit E.
      • Amat S.
      • et al.
      The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.
      • Holman D.B.
      • Yang W.
      • Alexander T.W.
      Antibiotic treatment in feedlot cattle: a longitudinal study of the effect of oxytetracycline and tulathromycin on the fecal and nasopharyngeal microbiota.
      By sampling the nasopharynx of preconditioned calves that were transported directly to a research feedlot, Holman and colleagues
      • Holman D.B.
      • Timsit E.
      • Amat S.
      • et al.
      The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.
      (2017) determined that presence and relative abundance of numerous bacteria changed significantly before (day 0) and after (day 2) transportation, with a higher abundance of Acinetobacter and Streptococcus and a lower abundance of Pasteurella and Bacillus after transportation. This shift in the nasopharyngeal microbiota occurred even in the absence of commingling or change in diet, because cattle were fed the same diet at both locations and were kept separate from other cattle housed at the feedlot.
      Adaptation to new diets and mixing cattle from multiple origins also alter the structure of the nasopharyngeal microbiota in cattle.
      • Hall J.A.
      • Isaiah A.
      • Estill C.T.
      • et al.
      Weaned beef calves fed selenium-biofortified alfalfa hay have an enriched nasal microbiota compared with healthy controls.
      ,
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      For example, recently weaned beef calves fed selenium-biofortified alfalfa hay had a higher bacterial diversity in their nasal cavities compared with healthy controls.
      • Hall J.A.
      • Isaiah A.
      • Estill C.T.
      • et al.
      Weaned beef calves fed selenium-biofortified alfalfa hay have an enriched nasal microbiota compared with healthy controls.
      Concerning the impact of mixing cattle on the nasopharyngeal microbiota, a minimum duration and frequency of contact among cattle is needed for horizontal transmission of commensal and pathogenic bacteria to occur. Commingling cattle for 24 hours at an auction market did not significantly affect the diversity or the composition of the nasopharyngeal bacteria in 2 groups of 15 recently weaned beef calves.
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      This suggests that being held for 24 hours at an auction market was not enough time to allow bacterial transfer or that the environment of the auction market was not conducive to interanimal bacterial transfer.
      Finally, parenteral antibiotics given at or soon after arrival to control BRD (ie, metaphylaxis) modifies diversity and composition of the nasopharyngeal microbiota.
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      ,
      • Holman D.B.
      • Yang W.
      • Alexander T.W.
      Antibiotic treatment in feedlot cattle: a longitudinal study of the effect of oxytetracycline and tulathromycin on the fecal and nasopharyngeal microbiota.
      ,
      • Holman D.B.
      • Timsit E.
      • Booker C.W.
      • et al.
      Injectable antimicrobials in commercial feedlot cattle and their effect on the nasopharyngeal microbiota and antimicrobial resistance.
      A single parenteral injection of either oxytetracycline or tulathromycin at feedlot placement altered the nasopharyngeal microbiota in comparison with cattle receiving only in-feed antibiotics for up to 60 days postadministration; oxytetracycline significantly reduced relative abundance of Mannheimia from feedlot entry to 60 days postarrival and cattle given either oxytetracycline or tulathromycin had a significantly lower relative abundance of Mycoplasma at day 60 compared with those given only an in-feed antibiotics.
      • Holman D.B.
      • Timsit E.
      • Booker C.W.
      • et al.
      Injectable antimicrobials in commercial feedlot cattle and their effect on the nasopharyngeal microbiota and antimicrobial resistance.
      Effects of parenteral oxytetracycline and tulathromycin on the nasopharyngeal microbiota were most important at days 2 and 5 post-treatment.
      • Holman D.B.
      • Yang W.
      • Alexander T.W.
      Antibiotic treatment in feedlot cattle: a longitudinal study of the effect of oxytetracycline and tulathromycin on the fecal and nasopharyngeal microbiota.
      At that time, both oxytetracycline and tulathromycin appeared to confer some protection against Pasteurella spp colonization in the nasopharynx.
      The nasopharyngeal microbiota of postweaned beef cattle often is dominated largely by Proteobacteria and Tenericutes, with lower proportions of Firmicutes, Actinobacteria, and Bacteroidetes (see Table 1). Of the dominant genera, Mycoplasma, Moraxella, Acinetobacter, Psychrobacter, Mannheimia, Pasteurella, and Corynebacterium are identified most frequently. There is considerable variability, however, in composition of the nasopharyngeal microbiota among groups of cattle and even among individual cattle within a group.
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      ,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.
      Furthermore, as discussed previously, the nasopharyngeal microbiota evolves significantly from entrance to the first weeks on feed. Therefore, it is difficult to identify a so-called normal microbiota in feedlot cattle.
      In summary, the nasopharyngeal microbiota of beef cattle changes significantly between weaning and the first weeks on feed. This evolution may explain why beef cattle are more susceptible to BRD during the first 40 days to 60 days on feed,
      • Babcock A.H.
      • Renter D.G.
      • White B.J.
      • et al.
      Temporal distributions of respiratory disease events within cohorts of feedlot cattle and associations with cattle health and performance indices.
      because an unstable microbiota is less resistant to colonization by pathogens.
      • Ducarmon Q.R.
      • Zwittink R.D.
      • Hornung B.V.H.
      • et al.
      Gut microbiota and colonization resistance against bacterial enteric infection.

      Composition of the Lower Respiratory Tract Microbiota

      The LRT, previously thought to be sterile, is now known to harbor a unique microbiota (Fig. 3).
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      ,
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      ,
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      ,
      • Amat S.
      • Alexander T.W.
      • Holman D.B.
      • et al.
      Intranasal bacterial therapeutics reduce colonization by the respiratory pathogen Mannheimia haemolytica in dairy calves.
      ,
      • Zeineldin M.M.
      • Lowe J.F.
      • Grimmer E.D.
      • et al.
      Relationship between nasopharyngeal and bronchoalveolar microbial communities in clinically healthy feedlot cattle.
      Characterization of the tracheal
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      ,
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      ,
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      and bronchial
      • Zeineldin M.M.
      • Lowe J.F.
      • Grimmer E.D.
      • et al.
      Relationship between nasopharyngeal and bronchoalveolar microbial communities in clinically healthy feedlot cattle.
      microbiotas in cattle revealed that these bacterial communities are distinct from nasal and nasopharyngeal microbiotas. Bacterial communities in the LRT are less rich and less diverse than the URT microbiotas,
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      ,
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      consistent with URT being directly exposed to ambient airborne microbial communities. Furthermore, some bacteria, such as Mycoplasma
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      ,
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      ,
      • Stroebel C.
      • Alexander T.
      • Workentine M.L.
      • et al.
      Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.
      and Pasteurella,
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      ,
      • Zeineldin M.M.
      • Lowe J.F.
      • Grimmer E.D.
      • et al.
      Relationship between nasopharyngeal and bronchoalveolar microbial communities in clinically healthy feedlot cattle.
      typically are enriched in the LRT compared with the URT.
      Figure thumbnail gr3
      Fig. 3Mean relative abundance of bacteria present at greater than or equal to 1% abundance at the genus level of different URT and LRT sampling locations in 15 healthy beef steer calves. URT, upper respiratory tract; LRT, lower respiratory tract.
      (From McMullen C, Alexander TW, Leguillette R, et al. Topography of the respiratory tract bacterial microbiota in feedlot beef calves. submitted for publication; with permission).
      Despite differences between URT and LRT microbiotas, most bacterial genera identified in the LRT also are present in the URT (McMullen C, Alexander TW, Leguillette R, et al. Topography of the respiratory tract bacterial microbiota in feedlot beef calves, submitted for publication).
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      ,
      • Zeineldin M.M.
      • Lowe J.F.
      • Grimmer E.D.
      • et al.
      Relationship between nasopharyngeal and bronchoalveolar microbial communities in clinically healthy feedlot cattle.
      This is explained by the fact that, in healthy animals, bacterial composition of the LRT is determined more by a constant flow (immigration and elimination) of transient bacteria originating from the URT than replication of resident bacteria.
      • Dickson R.P.
      • Erb-Downward J.R.
      • Freeman C.M.
      • et al.
      Bacterial topography of the healthy human lower respiratory tract.
      In humans, the bacteria reaching the lung primarily originate from the oropharynx and the mouth.
      • Dickson R.P.
      • Erb-Downward J.R.
      • Freeman C.M.
      • et al.
      Bacterial topography of the healthy human lower respiratory tract.
      In cattle, however, the nasopharynx seems to be the primary source of bacteria for the LRT. In a recent study by the authors’ team (McMullen C, Alexander TW, Leguillette R, et al. Topography of the respiratory tract bacterial microbiota in feedlot beef calves, submitted for publication), which compared bacterial communities of 17 locations across the respiratory tract, the lung microbiota was more compositionally similar to the nasopharynx than any other URT microbiota, including the mouth, oropharynx, palatine tonsils, or nostrils. Consequently, the nasopharyngeal microbiota should be the primary target for sampling strategies and the principal niche in the URT to modulate in order to promote good respiratory health (ie, prebiotics, probiotics, and bacteriophages).

      Influence of the bacterial microbiota on respiratory health

      Composition, diversity, and stability of the respiratory microbiota can play a role in either predisposing cattle to BRD or providing protection against colonization and/or proliferation of bacterial pathogens in the respiratory tract (also known as colonization resistance) (see Fig. 2).
      • Timsit E.
      • Holman D.B.
      • Hallewell J.
      • et al.
      The nasopharyngeal microbiota in feedlot cattle and its role in respiratory health.

      Role of the Composition of the Microbiota on Respiratory Health

      Primary bacteria involved in BRD are M haemolytica, P multocida, H somni, and Mycoplasma bovis (Table 2). Although these bacteria are opportunistic in nature, cattle with them in their respiratory tract are at higher risk of developing BRD.
      • Timsit E.
      • Hallewell J.
      • Booker C.
      • et al.
      Prevalence and antimicrobial susceptibility of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolated from the lower respiratory tract of healthy feedlot cattle and those diagnosed with bovine respiratory disease.
      For example, recently weaned beef cattle positive for M haemolytica on deep nasopharyngeal swabs at feedlot entry were more likely (odds ratio 1.7; 95% CI, 1.1–2.4) to be affected with BRD within 10 days after arrival than cattle negative for this bacterium.
      • Noyes N.R.
      • Benedict K.M.
      • Gow S.P.
      • et al.
      Mannheimia haemolytica in feedlot cattle: prevalence of recovery and associations with antimicrobial use, resistance, and health outcomes.
      Furthermore, in 174 dairy calves, relative abundance of Mannheimia and Mycoplasma was higher at days 14 and/or 28 in the nasopharynx of calves that subsequently developed BRD versus those that remained healthy.
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      Therefore, limiting colonization of opportunistic bacterial pathogens in the respiratory tract can reduce the prevalence of BRD in cattle.
      Table 2Changes in the upper and lower respiratory tract microbiota associated with bovine respiratory disease
      Data from Refs.
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      ,
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      ,
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      ,
      • Holman D.B.
      • McAllister T.A.
      • Topp E.
      • et al.
      The nasopharyngeal microbiota of feedlot cattle that develop bovine respiratory disease.
      ,
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      ,
      • Gaeta N.C.
      • Lima S.F.
      • Teixeira A.G.
      • et al.
      Deciphering upper respiratory tract microbiota complexity in healthy calves and calves that develop respiratory disease using shotgun metagenomics.
      ,
      • Zeineldin M.
      • Lowe J.
      • de Godoy M.
      • et al.
      Disparity in the nasopharyngeal microbiota between healthy cattle on feed, at entry processing and with respiratory disease.
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Evaluating the microbiome of two sampling locations in the nasal cavity of cattle with bovine respiratory disease complex (BRDC).
      • Johnston D.
      • Earley B.
      • Cormican P.
      • et al.
      Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue.
      ,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Comparison of the nasopharyngeal bacterial microbiota of beef calves raised without the use of antimicrobials between healthy calves and those diagnosed with bovine respiratory disease.
      ,
      • Klima C.L.
      • Holman D.B.
      • Ralston B.J.
      • et al.
      Lower respiratory tract microbiome and resistome of bovine respiratory disease mortalities.
      TypeStudy DesignSample TypeMain FindingsReference
      Postweaned feedlot beef calvesCross-sectional: 2 populations (dead calves with lung lesions at necropsy, n = 15; non-BRD related mortality, n = 3)BAL collected at necropsyM haemolytica, Mycoplasma bovis, and H somni were relatively abundant (>5%) in most but not all BRD samples. Other relatively abundant genera (>1%) included Acinetobacter, Bacillus, Bacteroides, Clostridium, Enterococcus, and Pseudomonas. Mycoplasma bovis was not detected in non-BRD lung samples.Klima et al,
      • Klima C.L.
      • Holman D.B.
      • Ralston B.J.
      • et al.
      Lower respiratory tract microbiome and resistome of bovine respiratory disease mortalities.
      2019
      Cross-sectional: 2 populations (BRD, n = 82; healthy pen-matched, n = 82)DNSBacterial communities differed between BRD and CTRL groups. Relative abundance of H somni, M haemolytica, Mycoplasma bovis, or P multocida did not differ between BRD and CTRL groups. The proportion of samples that contained Mycoplasma bovis was higher, however, in the BRD group (43.90%) compared with the CTRL group (18.29%). Richness was lower in cattle with BRD.McMullen et al,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Comparison of the nasopharyngeal bacterial microbiota of beef calves raised without the use of antimicrobials between healthy calves and those diagnosed with bovine respiratory disease.
      2019
      Cross-sectional over 5 wk after feedlot entry: 4 populations (calves sampled in 2015: BRD, n = 25 [5 pooled samples]; healthy, n = 30 [6 pooled samples]) and calves sampled in 2016: BRD, n = 8 [16 pooled samples]; healthy, n = 38 [10 pooled samples])NS and DNSBacterial communities differed between BRD and CTRL groups only in 2016 (not in 2015). In 2016, Psychrobacter was more abundant in calves with BRD compared with CTRL in weeks 4 and 5 after feedlot entry, whereas Moraxella was greater in calves with BRD compared with CTRL throughout all 5 wk after feedlot entry.McDaneld et al,
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Evaluating the microbiome of two sampling locations in the nasal cavity of cattle with bovine respiratory disease complex (BRDC).
      2018
      Cross-sectional: 2 populations (BRD, n = 60; healthy pen-matched, n = 60)DNS and TTABacterial communities present within the airways clustered into 4 distinct metacommunities that were associated with sampling locations and health status. Metacommunity 1, enriched with Mycoplasma bovis, M haemolytica, and P multocida, was dominant in the nasopharynx and trachea of cattle with BRD. In contrast, metacommunity 3, enriched with Mycoplasma dispar, Lactococcus lactis, and Lactobacillus casei, was present mostly in the trachea of CTRL cattle. Metacommunity 4, enriched with Corynebacterium, Jeotgalicoccus, Psychrobacter, and Planomicrobium, was present in the nasopharynx only. Metacommunity 2, enriched with H somni, Moraxella, and L lactis, was present in both BRD and CTRL cattle. Richness and diversity were lower in the trachea and nasopharynx of cattle with BRD.Timsit et al,
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      2018
      Longitudinal; 4 populations (BRD at entry, n = 22; BRD at diagnosis; n = 22; healthy at entry, n = 44; healthy at diagnosis, n = 10)DNSBacterial communities differed between BRD and CTRL groups. At the phylum level, Proteobacteria was higher in BRD calves vs CTRL (32.12% vs 16.32%). Actinobacteria (38.20% vs 16.58%) and Fusobacteria (3.86% vs 0.03%) were higher in CTRL. At the genus level, Acinetobacter (12.54% vs 2.16%), Solibacillus (3.71% vs 0.02%), and Pasteurella (2.38% vs 0.03%) were higher in BRD. Mycoplasma and Moraxella were numerically higher in BRD (but P > .05). Rathayibacter (20.09% vs 3.96%) was higher in CTRL. No difference in bacterial diversity and richness was observed between BRD and CTRL.Zeineldin et al,
      • Zeineldin M.
      • Lowe J.
      • de Godoy M.
      • et al.
      Disparity in the nasopharyngeal microbiota between healthy cattle on feed, at entry processing and with respiratory disease.
      2017
      Longitudinal: 2 populations of calves sampled at feedlot entry and on 60 d after entry (BRD, n = 5; healthy, n = 5)DNSBacterial communities differed between BRD-CTRL groups at d 0 and d 60. At the phylum level, abundance of Actinobacteria was lower in BRD cattle. At the family level, there was a greater relative abundance of Micrococcaceae (d 0), Lachnospiraceae (d 60), Lactobacillaceae (d 0), and Bacillaceae (d 0) in CTRL. Richness and diversity were lower in the nasopharynx of cattle with BRD at d 0 and d 60.Holman et al,
      • Holman D.B.
      • McAllister T.A.
      • Topp E.
      • et al.
      The nasopharyngeal microbiota of feedlot cattle that develop bovine respiratory disease.
      2015
      Post-weaned beef calves (not feedlot)Cross-sectional: 2 populations (BRD, n = 8; healthy, n = 11)DNS and TTANo difference in bacterial communities between BRD and CTRL groups.Nicola et al,
      • Nicola I.
      • Cerutti F.
      • Grego E.
      • et al.
      Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.
      2017
      Preweaned and post-weaned dairy calvesCross-sectional: 3 populations (clinical BRD with lung lesions at necropsy, n = 6; clinically healthy with lung lesions at necropsy, n = 12; clinically healthy without lung lesions at necropsy, n = 8)Lung tissue (cranial lung lobes) and lymph nodesLeptotrichiaceae, Fusobacterium, Mycoplasma, Trueperella, and Bacteroides had greater relative abundances in lung samples collected from fatal BRD cases, compared with clinically healthy calves without lung lesions. Leptotrichiaceae, Mycoplasma and Pasteurellaceae had higher relative abundances in lymph nodes collected from fatal BRD cases, compared with clinically healthy calves without lung lesions.Johnston et al,
      • Johnston D.
      • Earley B.
      • Cormican P.
      • et al.
      Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue.
      2017
      Preweaned dairy calvesLongitudinal: 1 population sampled at 14 d and 28 d of life. During the study period, calves had BRD (n = 6) or remained healthy (n = 10).DNSThe relative abundance of Pseudomonas fluorescens was higher in BRD calves at d 14 compared with CTRL. M haemolytica S1 and non-S1 were numerically higher at d 14 in calves with BRD compared with CTRL.Gaeta et al,
      • Gaeta N.C.
      • Lima S.F.
      • Teixeira A.G.
      • et al.
      Deciphering upper respiratory tract microbiota complexity in healthy calves and calves that develop respiratory disease using shotgun metagenomics.
      2017
      Longitudinal: 1 population sampled at 3 d, 14 d, 28 d, and 35 d of life. During the study period, calves had BRD (n = 37), otitis media (n = 62), or BRD and otitis media (n = 11) or remained healthy (n = 64).DNSAt d 3, heathy calves had significantly lower total bacterial loads in their nasopharynx than calves with BRD. The relative abundances of Mannheimia and Moraxella were higher in BRD calves at d 14. The relative abundance of Mannheimia and Mycoplasma were higher in BRD calves at d 28. Richness and diversity did not differ among groups.Lima et al,
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      2016
      Preweaned dairy calves and their damsLongitudinal; 1 population of calves sampled at 3 d, 14 d, and 35 d of age. During the study period, calves had BRD (n = 16), otitis media (n = 28), or BRD and otitis media (n = 5) or remained healthy (n = 32). Their dams were sampled just before calving.DNS and vaginal swabs (for the dams)The relative abundance of Mannheimia was significantly higher at d 14 in animals that eventually developed BRD than in calves that remained healthy. The genera Porphyromonas and Campylobacter were relatively more abundant in the vaginal microbiota of dams whose progeny developed disease, and Mannheimia and Caloramator were relatively more abundant in the vaginal microbiota of dams whose progeny remained healthy.Lima et al,
      • Lima S.F.
      • Bicalho M.L.S.
      • Bicalho R.C.
      The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.
      2019
      Abbreviations: BAL, bronchoalveolar lavage; CTRL, control; DNS, deep nasal swab (≥20 cm long); NS, nasal swab (<20 cm long); S1, serotype 1; TTA, transtracheal aspiration.
      Based on recent 16S rRNA sequencing, presence of bacteria other than Pasteurellaceae or Mycoplasma bovis in the respiratory tract also may predispose cattle to BRD (see Table 2). For example, Moraxella was enriched in the nasopharynx of calves that developed BRD.
      • Lima S.F.
      • Teixeira A.G.
      • Higgins C.H.
      • et al.
      The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.
      ,
      • Zeineldin M.
      • Lowe J.
      • de Godoy M.
      • et al.
      Disparity in the nasopharyngeal microbiota between healthy cattle on feed, at entry processing and with respiratory disease.
      ,
      • McDaneld T.G.
      • Kuehn L.A.
      • Keele J.W.
      Evaluating the microbiome of two sampling locations in the nasal cavity of cattle with bovine respiratory disease complex (BRDC).
      Furthermore, a bacterium from the Leptotrichiaceae family was more abundant among postmortem lung tissue samples from dairy calves that died from BRD compared with lesion-free lung tissue of clinically healthy calves.
      • Johnston D.
      • Earley B.
      • Cormican P.
      • et al.
      Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue.
      These findings indicate that there may be other bacterial species with the potential to be secondary BRD pathogens that the veterinary community is unaware of. Before implementing mitigation strategies against them, however, further research is needed to confirm a causal relation between their presence in the respiratory microbiota and BRD.
      Comparison of the respiratory tract microbiota of healthy calves with those that developed BRD revealed the presence of specific commensal bacteria in the respiratory tract that can confer protection against the disease (see Table 2). For example, cattle having a higher relative abundance of Lactobacillaceae and Bacillaceae in their nasopharynx at feedlot entry were less likely to develop BRD during the first 60 days on feed.
      • Holman D.B.
      • McAllister T.A.
      • Topp E.
      • et al.
      The nasopharyngeal microbiota of feedlot cattle that develop bovine respiratory disease.
      Furthermore, in a comparison of the nasopharyngeal and tracheal microbiotas of 60 feedlot cattle with BRD to 60 healthy pen-mates, tracheal microbiota of healthy cattle was enriched with Mycoplasma dispar, Lactococcus lactis, and Lactobacillus casei.
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      Commensal bacteria can confer resistance against colonization and proliferation of opportunistic bacterial pathogens through several mechanisms. First, resistance can be provided through occupation of an otherwise vacant respiratory niche. As a result, invading pathogens have to compete for adhesion receptors and nutrients. For example, numerous lactic acid bacteria (LAB) had greater adhesion to bovine bronchial epithelial cells than M haemolytica.
      • Amat S.
      • Subramanian S.
      • Timsit E.
      • et al.
      Probiotic bacteria inhibit the bovine respiratory pathogen Mannheimia haemolytica serotype 1 in vitro.
      Commensals in the nasopharynx also can directly inhibit growth of bacterial pathogens by modifying their environment (ie, production of lactic or acetic acid) or producing antimicrobial molecules (eg, bacteriocins and hydrogen peroxide).
      • Amat S.
      • Timsit E.
      • Baines D.
      • et al.
      Development of bacterial therapeutics against the bovine respiratory pathogen Mannheimia haemolytica.
      Finally, commensals can enhance colonization resistance against pathogens via immune stimulation of the host and modulation of mucosal inflammation. For example, Streptococcus salivarius inhibited inflammatory responses in human bronchial epithelial cells (ie, down-regulation of the nuclear factor κB pathway) and promoted host microbe homeostasis.
      • Cosseau C.
      • Devine D.A.
      • Dullaghan E.
      • et al.
      The commensal Streptococcus salivarius K12 downregulates the innate immune responses of human epithelial cells and promotes host-microbe homeostasis.
      New knowledge that commensal bacteria are not mere bystanders but have a role in maintaining respiratory health in cattle has led to advent of respiratory probiotics (discussed later).

      Role of the Overall Diversity and Stability of the Microbiota on Respiratory Health

      Because biodiversity correlates to efficiency of nutrient utilization by a community,
      • Cardinale B.J.
      • Duffy J.E.
      • Gonzalez A.
      • et al.
      Biodiversity loss and its impact on humanity.
      a more diverse bacterial community is, in theory, more likely to resist colonization by pathogens. For example, in children, colonization of the URT by acute otitis media pathogens (Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis) was associated with lower levels of diversity in the URT microbiota.
      • Pettigrew M.M.
      • Laufer A.S.
      • Gent J.F.
      • et al.
      Upper respiratory tract microbial communities, acute otitis media pathogens, and antibiotic use in healthy and sick children.
      In cattle, diversity of the URT and LRT was lower in cattle with BRD than in their healthy pen-mates.
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      ,
      • Holman D.B.
      • McAllister T.A.
      • Topp E.
      • et al.
      The nasopharyngeal microbiota of feedlot cattle that develop bovine respiratory disease.
      ,
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • et al.
      Comparison of the nasopharyngeal bacterial microbiota of beef calves raised without the use of antimicrobials between healthy calves and those diagnosed with bovine respiratory disease.
      Because overgrowth of pathogens in the respiratory tract could lead to a loss of diversity, however, it is difficult to determine whether reduced diversity predisposes cattle to BRD or is merely a consequence of proliferation of bacterial pathogens preceding clinical BRD. Therefore, additional longitudinal studies investigating the role of the microbiota diversity in respiratory health are needed.
      A lack of stability in the URT microbiota during the first year of life has been associated with an increased risk of URT disease (such as otitis media) in human infants.
      • Bosch A.
      • de Steenhuijsen Piters W.A.A.
      • van Houten M.A.
      • et al.
      Maturation of the infant respiratory microbiota, environmental drivers, and health consequences. a prospective cohort study.
      Perhaps disturbances in the bovine nasopharyngeal microbiota observed around the first month of age in dairy calves and during weaning/marketing in beef cattle predispose to colonization and/or proliferation of bacterial pathogens in the respiratory tract. Impact of microbiota stability on respiratory health in cattle, however, has not been reported.

      Modulation of the bacterial respiratory microbiota to promote health

      Currently, modulation of the respiratory microbiota to promote health is based primarily on the use of parenteral antimicrobials in cattle.
      • Nickell J.S.
      • White B.J.
      Metaphylactic antimicrobial therapy for bovine respiratory disease in stocker and feedlot cattle.
      This therapeutic strategy is effective in reducing URT colonization by M haemolytica or P multocida
      • Holman D.B.
      • Yang W.
      • Alexander T.W.
      Antibiotic treatment in feedlot cattle: a longitudinal study of the effect of oxytetracycline and tulathromycin on the fecal and nasopharyngeal microbiota.
      ,
      • Frank G.H.
      • Briggs R.E.
      • Loan R.W.
      • et al.
      Effects of tilmicosin treatment on Pasteurella haemolytica organisms in nasal secretion specimens of calves with respiratory tract disease.
      and thus typically decreases incidence of BRD for 2 weeks to 3 weeks after administration.
      • Nickell J.S.
      • White B.J.
      Metaphylactic antimicrobial therapy for bovine respiratory disease in stocker and feedlot cattle.
      Parenteral antimicrobials, however, also significantly disrupt microbial interactions among bacterial communities of the respiratory tract (Amat S, Timsit E, Workentine M, et al. Intranasal administration of bacterial therapeutics induces longitudinal modulation of the nasopharyngeal microbiota in post-weaned beef calves, submitted for publication). These communities then can become potentially more permissive to colonization by exogenous bacteria or proliferation of endogenous ones (Amat S, Timsit E, Workentine M, et al. Intranasal administration of bacterial therapeutics induces longitudinal modulation of the nasopharyngeal microbiota in post-weaned beef calves, submitted for publication). For example, on-arrival mass medication with tulathromycin was followed by rapid horizontal spread of a tulathromycin-resistant strain of M haemolytica.
      • Snyder E.
      • Credille B.
      • Berghaus R.
      • et al.
      Prevalence of multi drug antimicrobial resistance in isolated from high-risk stocker cattle at arrival and two weeks after processing.
      Furthermore, parenteral administration of antimicrobials such as tulathromycin or oxytetracycline increased abundance of resistant genes, such as erm(X), sul2, tet(M), msr(E), and tet(H), in the nasopharyngeal microbiota.
      • Holman D.B.
      • Yang W.
      • Alexander T.W.
      Antibiotic treatment in feedlot cattle: a longitudinal study of the effect of oxytetracycline and tulathromycin on the fecal and nasopharyngeal microbiota.
      ,
      • Holman D.B.
      • Timsit E.
      • Booker C.W.
      • et al.
      Injectable antimicrobials in commercial feedlot cattle and their effect on the nasopharyngeal microbiota and antimicrobial resistance.
      Fortunately, other approaches to modulate the respiratory microbiota and promote health (eg, probiotics, prebiotics, and bacteriophages) have potential as viable alternatives to parenteral antimicrobials.

      Definition, Mechanisms of Action, and Possible Application Route of Probiotics

      Probiotics are defined as “live microorganisms that when administered in adequate amounts, confer a health benefit to the host.”
      • Hill C.
      • Guarner F.
      • Reid G.
      • et al.
      Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic.
      It is noteworthy that a higher abundance in heathy animals compared with sick animals is not enough for a strain to be designated as probiotic. A causative relationship with health promoting effects also should be demonstrated.
      New probiotic postulates (based on Koch’s postulates) have been recently suggested in development of next-generation probiotics.
      • van den Broek M.F.L.
      • De Boeck I.
      • Kiekens F.
      • et al.
      Translating recent microbiome insights in otitis media into probiotic strategies.
      They are defined as follows:
      • 1.
        The microorganism is present in high abundance in healthy animals and decreased abundance in those suffering from a disease.
      • 3.
        The microorganism can be isolated from healthy animals and grown in pure culture.
      • 3.
        The cultured organism should promote health when introduced into a diseased animal.
      • 4.
        Because probiotics are, by definition, administered as live organisms, it should be possible to reisolate these microorganisms from the healthy experimental host and confirm that they are identical to the original specific causative agents.
      Based on these postulates, numerous LAB are potential candidates for next-generation probiotics.
      • Amat S.
      • Subramanian S.
      • Timsit E.
      • et al.
      Probiotic bacteria inhibit the bovine respiratory pathogen Mannheimia haemolytica serotype 1 in vitro.
      Probiotics promote respiratory health through 3 main mechanisms (Fig. 4).
      • van den Broek M.F.L.
      • De Boeck I.
      • Kiekens F.
      • et al.
      Translating recent microbiome insights in otitis media into probiotic strategies.
      First, they can have a direct antimicrobial action against bacterial respiratory pathogens by producing antimicrobial molecules, for example, lactic and acetic acid, bacteriocins, and hydrogen peroxide, in their microenvironment.
      • Amat S.
      • Timsit E.
      • Baines D.
      • et al.
      Development of bacterial therapeutics against the bovine respiratory pathogen Mannheimia haemolytica.
      Second, probiotics can enhance the epithelial barrier by, for example, stimulating production of mucin or antimicrobial peptides (ie, defensins, lysozymes, and cathelicidins).
      • Madsen K.
      • Cornish A.
      • Soper P.
      • et al.
      Probiotic bacteria enhance murine and human intestinal epithelial barrier function.
      ,
      • Bron P.A.
      • Kleerebezem M.
      • Brummer R.J.
      • et al.
      Can probiotics modulate human disease by impacting intestinal barrier function?.
      Finally, administration of probiotics also can modulate host immune responses (both innate and adaptative immunity) by interacting with host pattern recognition receptors of the mucosa.
      • Lebeer S.
      • Vanderleyden J.
      • De Keersmaecker S.C.
      Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens.
      For example, probiotic bacteria can modulate maturation of dendritic cells toward an anti-inflammatory interleukin (IL)-10 profile or stimulate regulatory T-cell activity to control overt inflammatory conditions.
      • Lebeer S.
      • Vanderleyden J.
      • De Keersmaecker S.C.
      Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens.
      In addition, they can modulate cytokine production and stimulate B-cell and antibody production (IgA and IgG).
      • Lebeer S.
      • Vanderleyden J.
      • De Keersmaecker S.C.
      Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens.
      Numerous other immunomodulatory effects of probiotics have been described, but a complete review of these mechanisms is outside the scope of this article (consult reviews by Lebeer and colleagues
      • Lebeer S.
      • Vanderleyden J.
      • De Keersmaecker S.C.
      Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens.
      [2010] and van den Broek and colleagues
      • van den Broek M.F.L.
      • De Boeck I.
      • Kiekens F.
      • et al.
      Translating recent microbiome insights in otitis media into probiotic strategies.
      [2019] for additional information). Most reports on effects of probiotics on host immunity are from humans or mice and not cattle.
      Figure thumbnail gr4
      Fig. 4Postulated beneficial modes of action of respiratory probiotics: (1) competition with pathogens for nutrients, adhesion sites, and receptors; (2) production of antimicrobial molecules, such as lactic acid, bacteriocins, and hydrogen peroxide (H2O2); (3) stimulation of epithelial cells to modulate mucin and antimicrobial peptide production; (4) modulation of the immune system via antigen-presenting cells (APCs); (5) modulation of cytokine production; (6) stimulation of increased B-cell production; and (7) stimulation of antibody production (IgA and IgG). Th1/2, T helper 1/2 cells; Treg, regulatory T cells.
      (Adapted from van den Broek MFL, De Boeck I, Kiekens F, Boudewyns A, Vanderveken OM, Lebeer S. Translating recent microbiome insights in otitis media into probiotic strategies. Clin Microbiol Rev. 2019; 3, 32; with permission.)
      Traditionally, probiotics have been administered by an oral route. Orally applied probiotics could benefit the URT via systemic immune effects.
      • Budden K.F.
      • Gellatly S.L.
      • Wood D.L.
      • et al.
      Emerging pathogenic links between microbiota and the gut-lung axis.
      They do not, however, have a direct antimicrobial action against bacterial respiratory pathogens, and they do not affect the URT’s local immune response. Conversely, nasal application of probiotics has the advantage of promoting more direct contact of the applied organisms with the respiratory tract mucosa and microbiota.
      • van den Broek M.F.L.
      • De Boeck I.
      • Kiekens F.
      • et al.
      Translating recent microbiome insights in otitis media into probiotic strategies.
      Furthermore, by using the nasal route, probiotics do not have to survive transit through the gastrointestinal tract (especially the rumen). Therefore, the next logical step is to design probiotics that can be applied intranasally.

      Intranasal Administration of Probiotics in Cattle

      To investigate whether nasal application of probiotics can modulate the respiratory microbiota to promote health in cattle, the authors’ team first selected in vitro probiotic strains originating from the nasopharynx of healthy cattle with properties that are important for URT probiotics (discussed later).
      • Amat S.
      • Timsit E.
      • Baines D.
      • et al.
      Development of bacterial therapeutics against the bovine respiratory pathogen Mannheimia haemolytica.
      Then, these probiotics strains were administered to dairy calves
      • Amat S.
      • Alexander T.W.
      • Holman D.B.
      • et al.
      Intranasal bacterial therapeutics reduce colonization by the respiratory pathogen Mannheimia haemolytica in dairy calves.
      and beef calves (Amat S, Timsit E, Workentine M, et al. Intranasal administration of bacterial therapeutics induces longitudinal modulation of the nasopharyngeal microbiota in post-weaned beef calves, submitted for publication) to investigate their health-promoting effects.
      For selection of probiotic strains, the authors used a stepwise approach.
      • Amat S.
      • Timsit E.
      • Baines D.
      • et al.
      Development of bacterial therapeutics against the bovine respiratory pathogen Mannheimia haemolytica.
      Bacteria isolated from the nasopharynx of healthy cattle for their ability to inhibit M haemolytica (178 isolates from 12 genera). Subsequently, abilities of selected isolates were evaluated to adhere to bovine turbinate (BT) cells (n = 47), compete against M haemolytica for BT cell adherence (n = 15), and modulate gene expression in BT cells (n = 10). Lactobacillus strains had the strongest inhibition against M haemolytica, with 88% of isolates having inhibition zones ranging from 17 mm to 23 mm. All isolates tested in competition assays reduced M haemolytica adherence to BT cells (32% to 78%). Among the 84 bovine genes evaluated, selected isolates slightly upregulated expression of IL-8 and IL-6. After ranking isolates for greatest inhibition, adhesion, competition, and immunomodulation properties, 6 Lactobacillus strains from 4 different species were selected as the best URT probiotic candidates: L amylovorous, L buchneri (2 strains), L curvatus, and L paracasei (2 strains). The authors primarily focused on LAB because these bacteria have a long history of safe use (eg, generally recognized as safe). Other bacteria, however, such as Mycoplasma dispar, also could have some probiotic properties, because they were present in higher abundance in healthy animals versus sick animals.
      • Timsit E.
      • Workentine M.
      • van der Meer F.
      • et al.
      Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia.
      Health-promoting effects of the 6 selected Lactobacillus strains were first evaluated in dairy calves.
      • Amat S.
      • Alexander T.W.
      • Holman D.B.
      • et al.
      Intranasal bacterial therapeutics reduce colonization by the respiratory pathogen Mannheimia haemolytica in dairy calves.
      For this evaluation, 1-week-old to 3-week-old dairy calves received either an intranasal cocktail of the 6 probiotic strains (3 × 109 colony-forming units [CFUs] per strain; n = 12) 24 hours prior to an intranasal M haemolytica challenge (3 × 108 CFUs), or only phosphate buffered saline (PBS) prior to challenge (control group; n = 12). Nasal swabs were collected over the course of 16 days after probiotic inoculation. Probiotic strains were reisolated up to 13 days after inoculation, with variation existing among strains and calves. Their administration significantly reduced nasal colonization by M haemolytica. It also modified composition and reduced the diversity of the nasal microbiota and altered interbacterial relationships among the 10 most abundant genera. This study demonstrated, for the first time, that intranasal probiotics developed from bovine nasopharyngeal Lactobacillus could reduce nasal colonization by M haemolytica in dairy calves.
      In a second study, the authors investigated the health promoting effects of the same probiotic cocktail in beef cattle (Amat S, Timsit E, Workentine M, et al. Intranasal administration of bacterial therapeutics induces longitudinal modulation of the nasopharyngeal microbiota in post-weaned beef calves, submitted for publication). In that study, on arrival at the feedlot, newly received beef steers either received (1) an intranasal cocktail of the 6 strains (3 × 109 CFUs per strain; n = 20); (2) intranasal PBS (negative control; n = 20); or (3) parenteral tulathromycin (Draxxin [Zoetis (Kirkland, Ontario, Canada)]) (positive control; 2.5 mg/kg; n = 20). Nasopharyngeal swabs were collected for up to 42 days post-treatment. Nasopharyngeal colonization by probiotics was most apparent at day 2 postinoculation; however, administration of probiotics modified composition and reduced the diversity of the nasopharyngeal microbiota for up to 42 days. Compared with PBS and probiotics, parenteral tulathromycin decreased bacterial load in the nasopharynx and increased abundance of the antibiotic-resistant gene msr(E). There were no significant effects among treatments on relative abundance of M haemolytica, P multocida, or H somni. This second study demonstrated that a unique intranasal inoculation of probiotics could modify the nasopharyngeal microbiota for up to 42 days in postweaned beef cattle. Unfortunately, it did not provide useful information on potential health promoting effects of the probiotic cocktail, because the disease challenge was very low, with only 5 of the 60 calves diagnosed with BRD during the study period (3 in the PBS group and 2 in the probiotic group).
      In summary, a single intranasal administration of 6 selected Lactobacillus strains modified the nasopharyngeal microbiota of dairy cattle and beef cattle and provided colonization resistance against M haemolytica. Additional research is needed to define the optimal dose and duration of application of this probiotic cocktail to maximize health benefits as well as to further confirm its health promoting effect (ie, can it reduce incidence of BRD after administration, which is the outcome of interest for the cattle industry). Perhaps a single inoculation is not enough to prevent BRD. In human studies, intranasal probiotics to prevent otitis media in infants typically are given multiples times over a few days or weeks; for example, 10 days per month over 2 consecutive months or twice daily for 10 days.
      • van den Broek M.F.L.
      • De Boeck I.
      • Kiekens F.
      • et al.
      Translating recent microbiome insights in otitis media into probiotic strategies.
      Furthermore, it is noteworthy that probiotics have different abilities to colonize and influence a particular individual. For example, it has been reported that transient colonization by probiotic strains is highly variable in the lower gastrointestinal tract of humans, with some humans being more permissive to colonization and others being resistant.
      • Zmora N.
      • Zilberman-Schapira G.
      • Suez J.
      • et al.
      Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features.
      It, therefore, is possible that the composition of the probiotic cocktail should be adapted to animal age and production system, that is, preweaned dairy calves, preweaned or postweaned beef cattle, and veal calves.

      Other Strategies: Bacteriophages and Prebiotics

      Bacteriophage therapy is another way to modulate the bacterial structure of the respiratory microbiota.
      • Wienhold S.M.
      • Lienau J.
      • Witzenrath M.
      Towards inhaled phage therapy in Western Europe.
      Bacteriophages are viruses that infect bacteria. They either can be stably integrated into bacterial genomes (lysogenic phase) or can replicate and lyse bacteria, releasing virus particles (lytic phase).
      • Kutateladze M.
      • Adamia R.
      Bacteriophages as potential new therapeutics to replace or supplement antibiotics.
      Bacteriophages are highly specific and typically infect only 1 bacterial species, serotype, or strain. Their inoculation in the nasopharynx of cattle, therefore, can remove bacterial respiratory pathogens without having an impact on the commensal flora.
      • Wienhold S.M.
      • Lienau J.
      • Witzenrath M.
      Towards inhaled phage therapy in Western Europe.
      Furthermore, bacteriophages can amplify exponentially after administration and thus do not always need multiple administrations. Bacteriophages with lytic properties against M haemolytica have been isolated and characterized.
      • Urban-Chmiel R.
      • Wernicki A.
      • Stegierska D.
      • et al.
      Isolation and characterization of lytic properties of bacteriophages specific for M. haemolytica strains.
      Unfortunately, to the authors’ best knowledge, there are no published data on their use to remove M haemolytica from the nasopharynx of cattle. The increasing prevalence of antibiotic resistance in M haemolytica and P multocida isolated in the United States,
      • Snyder E.
      • Credille B.
      • Berghaus R.
      • et al.
      Prevalence of multi drug antimicrobial resistance in isolated from high-risk stocker cattle at arrival and two weeks after processing.
      ,
      • Woolums A.R.
      • Karisch B.B.
      • Frye J.G.
      • et al.
      Multidrug resistant Mannheimia haemolytica isolated from high-risk beef stocker cattle after antimicrobial metaphylaxis and treatment for bovine respiratory disease.
      Canada,
      • Timsit E.
      • Hallewell J.
      • Booker C.
      • et al.
      Prevalence and antimicrobial susceptibility of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolated from the lower respiratory tract of healthy feedlot cattle and those diagnosed with bovine respiratory disease.
      and Europe
      • Schonecker L.
      • Schnyder P.
      • Overesch G.
      • et al.
      Associations between antimicrobial treatment modalities and antimicrobial susceptibility in Pasteurellaceae and E. coli isolated from veal calves under field conditions.
      nevertheless is creating an impetus to further investigate bacteriophage therapies in cattle.
      Prebiotics are nonviable substrates that serve as nutrients for beneficial microorganisms harbored by the host, including administered probiotic strains and indigenous (resident) microorganisms.
      • Gibson G.R.
      • Hutkins R.
      • Sanders M.E.
      • et al.
      Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics.
      Commonly studied prebiotics include fructo-oligosaccharides, galacto-oligosaccharides, inulin, and resistant starch.
      • Vitetta L.
      • Vitetta G.
      • Hall S.
      Immunological tolerance and function: associations between intestinal bacteria, probiotics, prebiotics, and phages.
      Administration of prebiotics alone or in combination with probiotics in the URTs of cattle could promote the selective growth of bacteria considered beneficial, for example, Lactobacilli. Unfortunately, to date, prebiotics have been used solely to selectively enhance the growth of beneficial bacteria in the digestive tract, and further research is needed before recommending their use for modulating the respiratory tract microbiota of cattle.

      Summary

      The respiratory tract of cattle is colonized by complex bacterial ecosystems also known as bacterial microbiotas. These microbiotas evolve over time and are shaped by numerous factors, including maternal vaginal microbiota, environment, age, diet, parenteral antimicrobials, and stressful events (eg, transportation and commingling). The resulting microbiota can be diverse and enriched with known beneficial bacteria (ie, Lactobacillus and Lactococcus) that can provide colonization resistance against opportunistic bacterial pathogens or, on the contrary, with bacterial pathogens, such as M haemolytica, P multocida, H somni, or Mycoplasma bovis, predisposing cattle to respiratory disease. Beneficial bacteria promote health through 3 main mechanisms: (1) direct antimicrobial action against bacterial pathogens, (2) enhancement of the epithelial barrier, and (3) modulation of the host immune response. Among beneficial bacteria, Lactobacillus are of particular interest because they generally are regarded as safe. Intranasal inoculation of a cocktail of 6 Lactobacillus strain modified the structure of the nasopharyngeal microbiota over a few weeks in beef calves and dairy calves and provided colonization resistance against M haemolytica. That the respiratory microbiota can be modulated by nonantimicrobial approaches to promote health creates new potential strategies for prevention and treatment of BRD.

      Acknowledgments

      The authors gratefully acknowledge Dr. John Kastelic for editing the article.

      Disclosure

      Dr E. Timsit is an Innovation Scientist at Ceva Animal Health and is responsible for early phases of drug discovery and development. None of the authors of this article has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the article. This article reflects the views of the authors and should not be construed as representing the views of Ceva Animal Health.

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