Introduction
Multiple factors have been associated with the development of bovine respiratory disease (BRD), but bacterial species, including Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis, and Pasteurella multocida, are frequently implicated (Confer, Reference Confer2009). The upper respiratory tract is a reservoir of these opportunistic pathogens, which can proliferate and infect the lungs when cattle immunity is compromised due to stress or primary viral infections (Hodgson et al., Reference Hodgson, Aich, Hokamp, Roche, Brinkman, Potter, Babiuk and Griebel2005). High-risk cattle populations entering feedlots are most susceptible to BRD, and as a result, these cattle are often administered metaphylactic antimicrobials to prevent infections after feedlot placement. However, there are public and scientific concerns regarding antimicrobial use in livestock production (Cameron and McAllister, Reference Cameron and McAllister2016), and recent reports indicate high levels of resistance in important BRD pathogens from feedlot cattle, potentially limiting the antimicrobials available for treatment (Portis et al., Reference Portis, Lindeman, Johansen and Stoltman2012; Anholt et al., Reference Anholt, Klima, Allan, Matheson-Bird, Schatz, Ajitkumar, Otto, Peters, Schmid, Olson, McAllister and Ralston2017; Timsit et al., Reference Timsit, Hallewell, Booker, Tison, Amat and Alexander2017). Thus, novel methods to reduce antimicrobial use and mitigate BRD-related pathogenic bacteria are needed.
While pathogenic bacteria that can be cultured in the laboratory have been the main focus of research on the bovine respiratory tract, advances and affordability of next-generation sequencing have led to an increased number of studies regarding the respiratory microbiome. Resulting from these studies is an improved understanding of the importance of the mammalian microbiome in relation to host health, and it is clear that the resident microbiota of the respiratory tract has a critical role in preventing colonization of pathogens (Bogaert et al., Reference Bogaert, De Groot and Hermans2004; Cho and Blaser, Reference Cho and Blaser2012). Establishment and stability of the respiratory microbiota are important for homeostasis while disruption can predispose to pathogenesis (Man et al., Reference Man, de Steenhuijsen Piters and Bogaert2017). For example, reductions in bacterial community density and diversity following antimicrobial treatment in humans have been associated with an increased risk of overgrowth of bacterial pathogens and establishment of respiratory diseases (Pettigrew et al., Reference Pettigrew, Laufer, Gent, Kong, Fennie and Metlay2012). There is evidence showing that the cattle respiratory microbiota are also susceptible to disturbances. In beef cattle, transportation to a feedlot (Holman et al., Reference Holman, Timsit, Amat, Abbott, Buret and Alexander2017), diet composition (Hall et al., Reference Hall, Isaiah, Estill, Pirelli and Suchodolski2017), and antimicrobial administration (Holman et al., Reference Holman, Timsit, Booker and Alexander2018; Holman et al., Reference Holman, Yang and Alexander2019) have previously been shown to alter the diversity and structure of the nasopharyngeal microbiota, highlighting that respiratory bacteria of cattle are affected by industry management practices. Bacterial diversity and richness were reduced in the nasopharynx of cattle that developed BRD early in the feeding period compared with cattle that remained healthy (Holman et al., Reference Holman, McAllister, Topp, Wright and Alexander2015a) and certain bacterial communities have been associated with bovine respiratory health (Timsit et al., Reference Timsit, Workentine, van der Meer and Alexander2018). These data suggest that distinct microbial profiles of the respiratory tract could be used to improve BRD diagnosis. In addition, microbiota-based interventions (e.g. bacterial therapeutics) may provide new opportunities for managing BRD.
Structure and composition of the bovine respiratory microbiota
Microbiota studies based on short (<400 bp) 16S rRNA gene sequences from beef cattle nasopharyngeal (NP) swabs, trans-tracheal aspirations (TTA) and bronchoalveolar lavages (BAL) have revealed that mucosal surfaces of the upper and lower airways are colonized by diverse microbial communities. The NP microbiota of feedlot cattle has been more extensively characterized, compared to the lower respiratory microbiota, and has been shown to be comprised of at least 29 different phyla and 300 different genera (Timsit et al., Reference Timsit, Workentine, Schryvers, Holman, van der Meer and Alexander2016a). Although the proportions of bacteria vary among individual cattle, and also the time of sampling during production, the most commonly identified phyla within the NP microbiota include Proteobacteria, Firmicutes, Actinobacteria, Bacteriodetes, and Tenericutes, accounting for more than 90% of 16S rRNA sequences (Holman et al., Reference Holman, Timsit and Alexander2015b, Reference Holman, Timsit, Amat, Abbott, Buret and Alexander2017; Zeineldin et al., Reference Zeineldin, Lowe, de Godoy, Maradiaga, Ramirez, Ghanem, Abd El-Raof and Aldridge2017a; Timsit et al., Reference Timsit, Workentine, van der Meer and Alexander2018). Within these phyla, Corynebacterium, Moraxella, Mycoplasma, Pasteurella, Mannheimia, Psychrobacter, and Staphylococcus are the most relatively abundant NP genera. Overall, TTA and BAL samples from feedlot cattle have shown that the microbiota of the lungs have similar taxonomic profiles compared to the upper respiratory tract, but can have reduced diversity and geographic-specific abundances of certain bacteria (Zeineldin et al., Reference Zeineldin, Lowe, Grimmer, de Godoy, Ghanem, Abd El-Raof and Aldridge2017b; Timsit et al., Reference Timsit, Workentine, van der Meer and Alexander2018). This may be in part due to physiological differences between the upper and lower respiratory tract, including variations in pH, relative humidity, temperature, and partial pressure of oxygen and carbon dioxide (Man et al., Reference Man, de Steenhuijsen Piters and Bogaert2017). Although methods using short 16S rRNA sequencing have provided tremendous opportunities for bacterial microbiota analysis, taxonomic discrimination can be limited. Using shotgun metagenomics (Gaeta et al., Reference Gaeta, Lima, Teixeira, Ganda, Oikonomou, Gregory and Bicalho2017) or long-read sequencing technologies may be useful to further categorize species and subspecies differences in respiratory tract bacteria in future studies.
Management factors influencing the bovine respiratory microbiota
Several studies have shown that the NP microbiota of beef calves changes after transportation to a feedlot. In one study that sampled the nasopharynx of calves at weaning, upon feedlot arrival, and 40 days after feedlot placement, it was shown that the NP microbiota underwent a profound evolution, with the abundance of 92 operational taxonomic units (OTUs) significantly changing over time (Timsit et al., Reference Timsit, Workentine, Schryvers, Holman, van der Meer and Alexander2016a). In a subsequent study that focused on shorter time points after feedlot placement, the structure and composition of the NP microbiota were observed to change within 2 days of feedlot placement, increasing in both phylogenetic diversity and richness (Holman et al., Reference Holman, Timsit, Amat, Abbott, Buret and Alexander2017). In both those studies by Timsit et al. (Reference Timsit, Workentine, Schryvers, Holman, van der Meer and Alexander2016a) and Holman et al. (Reference Holman, Timsit, Amat, Abbott, Buret and Alexander2017), cattle were not administered antimicrobials or implants which could have biased the results. Interestingly, transportation to, and commingling at, an auction market for 24 h did not significantly influence NP or tracheal bacterial communities in recently weaned beef calves (Stroebel et al., Reference Stroebel, Alexander, Workentine and Timsit2018) indicating that feedlot introduction has a strong influence on shaping the respiratory microbiota throughout the beef continuum. It should be noted however that both farm of origin and feedlot practice can influence the pattern of respiratory microbiota evolution (McMullen et al., Reference McMullen, Orsel, Alexander, van der Meer, Plastow and Timsit2018; Stroebel et al., Reference Stroebel, Alexander, Workentine and Timsit2018), thus the changes observed in cattle are not necessarily common. The instability in respiratory microbiota observed after feedlot placement might contribute to why cattle are most likely to be affected with BRD during the first weeks after weaning and arrival at a feedlot. Factors that may lead to changes in the respiratory microbiota may include a reduction in calf immunity due to stress from weaning and transportation, and colonization by bacteria originating from the feedlot environment or new pen mates (Timsit et al., Reference Timsit, Holman, Hallewell and Alexander2016b). In addition, diet transition before or after feedlot placement may influence the respiratory microbiota (Hall et al., Reference Hall, Isaiah, Estill, Pirelli and Suchodolski2017).
Administration of antimicrobials can also affect the microbiota by inhibiting the growth of certain bacteria, and potentially promoting the growth of bacteria that have intrinsic or acquired resistance to the antimicrobial. In children, antibiotic use has been linked to an altered microbial community structure in the upper respiratory tract for up to 6 months after administration (Pettigrew et al., Reference Pettigrew, Laufer, Gent, Kong, Fennie and Metlay2012), indicating that a prolonged effect takes place. Recently, it was observed that alterations in the NP microbiota of commercial cattle were apparent 60 days after injection with either oxytetracycline or tulathromycin (Holman et al., Reference Holman, Timsit, Booker and Alexander2018). In a controlled study analyzing the effects of these same two antimicrobials on the NP microbiota across 34 days, perturbation of the NP microbiota was greatest 2 and 5 days after administration (Holman et al., Reference Holman, Yang and Alexander2019). In the study by Holman et al. (Reference Holman, Yang and Alexander2019), it took 12 days for the NP microbiota to recover after tulathromycin injection, whereas recovery was not apparent after 34 days for the oxytetracycline-treated cattle. Interestingly, shortly after administration (2–5 days), both antimicrobials reduced the abundance of Pasteurella spp.; however, one Mycoplasma OTU was enriched in oxytetracycline-treated cattle at the end of the study (d 34). Although it was not known whether the single Mycoplasma OTU belonged to a pathogenic species, these studies show that administration of antimicrobials to cattle can have short- and long-term impacts on the NP microbiota.
The role of the microbiota in respiratory health
An increasing number of studies suggest that the bovine respiratory microbiota play an important role in defining respiratory health and disease in cattle (Timsit et al., Reference Timsit, Workentine, Schryvers, Holman, van der Meer and Alexander2016a; Zeineldin et al., Reference Zeineldin, Lowe and Aldridge2019). Evidence of bacterial competition within the bovine respiratory tract was first shown by Corbeil et al., when they observed that bacteria from the nasopharynx either enhanced or limited in vitro growth of the BRD pathogens M. haemolytica, P. multocida, and H. somni (Corbeil et al., Reference Corbeil, Woodward, Ward, Mickelsen and Paisley1985). Enhancing bacteria included Micrococcus, Staphylococcus, Corynebacterium, Rhodococcus, Moraxella, and Actinobacter isolates, while isolates of Bacillus were the strongest inhibitors. Interestingly, it was subsequently observed that cattle sampled by deep nasal swabs at feedlot entry, and later developed BRD during the feeding period, had lower proportions of NP Bacillaceae and Lactobacillaceae family members compared to cattle that remained healthy (Holman et al., Reference Holman, McAllister, Topp, Wright and Alexander2015a). Thus, members of these bacterial families may provide colonization resistance against BRD bacterial pathogens. In support of this, a case–control study compared the NP and TTA microbiota collected from feedlot calves diagnosed with BRD and healthy pen mates (Timsit et al., Reference Timsit, Workentine, van der Meer and Alexander2018). In that study, distinct bacterial metacommunities were observed to be associated with the upper or lower respiratory tract, as well as BRD status. One metacommunity included Lactococcus lactis and Lactobacillus casei, and was mostly associated with the trachea of healthy calves. Overall, these studies highlight that differences in respiratory microbiota have been associated with BRD status in cattle. Given these observed associations, microbiota-based applications could potentially be used for both diagnosis and prevention of BRD.
Microbiota-based diagnostics for BRD
The development of microbiota-based diagnostics stems from in-depth analyses of microbial composition within a host and associations observed between specific microbial community members and a host phenotype. Microbiota-based diagnostics have been studied for gastrointestinal applications, including inflammatory bowel disease (Eck et al., Reference Eck, de Groot, de Meij, Welling, Savelkoul and Budding2017) and gut infections (Raes, Reference Raes2016). Recently, Langelier et al. (Reference Langelier, KL, Moazed, MR, ED, Deiss, Belzer, Bolourchi, Caldera, Fung, Jauregui, Malcolm, Lyden, Khan, Vessel, Quan, Zinter, CY, ED, Wilson, Miller, MA, KS, Christenson, CS and JL2018) developed a metagenomic sequencing-based method that simultaneously evaluated three core elements of acute airway infections, including the pathogen, airway microbiome and host response. The method allowed for accurate diagnosis of lower respiratory tract infections in critically ill adults. Given that BRD-affected cattle have been shown to have different microbiota profiles compared to healthy pen mates (Holman et al., Reference Holman, McAllister, Topp, Wright and Alexander2015a; Zeineldin et al., Reference Zeineldin, Lowe, de Godoy, Maradiaga, Ramirez, Ghanem, Abd El-Raof and Aldridge2017a) and communities have been identified that were associated with BRD phenotype (Timsit et al., Reference Timsit, Workentine, van der Meer and Alexander2018), it may be possible to use microbiota data to aid in the diagnosis of BRD. However, complications may arise from the polymicrobial nature of BRD infections and the fact that cattle source and management practices can alter respiratory bacteria. Therefore, further studies are needed to characterize the microbiotas of beef cattle before realizing their full potential for BRD diagnosis. Application of additional ‘omics’ technologies to evaluate host–pathogen interactions during BRD, such as transcriptomics or metabolomics, will help to better define the respiratory microbiota for use in diagnostics.
Bacterial therapeutics to mitigate BRD pathogens
Research on probiotics has significantly increased over the last 10 years, and has coincided with improved methods to culture bacteria and reduced costs of massive parallel sequencing technologies to analyze microbiota. These technologies have allowed in-depth study of host metagenomics and are providing important information on bacteria associated with certain phenotypes, such as diseases like BRD. There are ongoing efforts to identify strains of bacteria within a host's microbiota and developing them into probiotics that could potentially alter a phenotype after administration or prevent infections by pathogens. Strains of this nature however likely fall outside of the traditionally defined probiotics (e.g. Lactobacillus, Bifidobacterium) and would be used for therapeutic purposes. As such, they have been coined ‘next-generation probiotics’ (Patel and DuPont, Reference Patel and DuPont2015), or bacterial therapeutics. Their development employs a targeted approach combining microbiota sequencing and culturing, and is promising for tailored biotherapeutics that can be used as alternatives to antimicrobials in livestock. Recently, it was shown that commercial probiotic bacterial strains were capable of colonizing bovine bronchial epithelial cells and inhibiting M. haemolytica in vitro (Amat et al., Reference Amat, Subramanian, Timsit and Alexander2017). In a follow-up study, the same authors utilized microbiota data from cattle, and a step-wise targeted approach, to develop bacterial therapeutics against M. haemolytica that originated from the respiratory tract of feedlot calves (Amat et al., Reference Amat, Timsit, Baines, Yanke and Alexander2019). Intranasal application of six therapeutic candidate strains was capable of reducing colonization by M. haemolytica in challenged calves, showing that bacteria sourced from the bovine respiratory tract may have utility in reducing the proliferation of BRD pathogens (Amat et al., Reference Amat, Alexander, Holman, Schwinghamer and Timsit2020).
Conclusion
While the bovine respiratory microbiota is dynamic and can be altered by a number of management practices, it is not yet clear how perturbations directly impact BRD development. However, evidence exists to show that the respiratory microbiota contributes to cattle health potentially by providing colonization resistance against pathogens. A better understanding of how the respiratory microbiota relates to susceptibility and progression of BRD, and in maintaining respiratory health, may lead to the development of new technologies for diagnosing and mitigating BRD in feedlot cattle.
Acknowledgements
The research conducted by TWA is supported by grants from Alberta Agriculture and Forestry, Agriculture and Agri-Food Canada, and the Beef Cattle Research Council.
