Prevalence and antimicrobial susceptibility of Mycoplasma bovis from the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bovine respiratory disease.

Mycoplasma bovis is an important respiratory pathogen of cattle. In this study, the prevalence and antimicrobial susceptibility of M. bovis were evaluated from two Cohorts of feedlot cattle spanning an 8-year period. In the first study conducted in 2008-2009, nasopharyngeal swabs from cattle sampled at feedlot entry and after 60 days on feed were collected (Cohort 1). In a second study conducted in 2015-2016, nasopharyngeal and trans-tracheal samples were collected from cattle diagnosed with bovine respiratory disease (BRD) and matching healthy controls (Cohort 2). For Cohort 1, the prevalence of M. bovis was lower in cattle at entry compared to when the same individuals were sampled ≥60 days later (P < 0.05). For Cohort 2, the prevalence of M. bovis was greater in both nasopharyngeal and tracheal samples from cattle diagnosed with BRD, compared to controls (P < 0.05). In both Cohorts, almost all isolates were resistant to tilmicosin. Compared to M. bovis from Cohort 1, isolates of Cohort 2 exhibited increased resistance to clindamycin, enrofloxacin, florfenicol, tylosin, and tulathromycin, with the latter showing resistance levels >90 %. These data suggest that antimicrobials used to prevent and treat BRD selected for resistance in M. bovis over the 8-year period. For macrolides, cross-resistance occurred and M. bovis can retain resistance even when antimicrobial selection pressure is removed. Within 9 years of commercial availability of tulathromycin, the majority of M. bovis displayed resistance. Therefore, longitudinal evaluation of resistance in respiratory pathogens is important to ensure efficacious treatment of BRD.

A Single Intranasal Dose of Bacterial Therapeutics to Calves Confers Longitudinal Modulation of the Nasopharyngeal Microbiota: a Pilot Study.

To address the emergence of antimicrobial-resistant pathogens in livestock, microbiome-based strategies are increasingly being sought to reduce antimicrobial use. Here, we describe the effects of intranasal application of bacterial therapeutics (BTs) on the bovine respiratory microbiota and used structural equation modeling to investigate the causal networks after BT application. Beef cattle received (i) an intranasal cocktail of previously characterized BT strains, (ii) an injection of metaphylactic antimicrobial (tulathromycin), or (iii) intranasal saline. Despite being transient colonizers, inoculated BT strains induced longitudinal modulation of the nasopharyngeal bacterial microbiota while showing no adverse effect on animal health. The BT-mediated changes in bacteria included reduced diversity and richness and strengthened cooperative and competitive interactions. In contrast, tulathromycin increased bacterial diversity and antibiotic resistance and disrupted bacterial interactions. Overall, a single intranasal dose of BTs can modulate the bovine respiratory microbiota, highlighting that microbiome-based strategies have potential in being utilized to mitigate bovine respiratory disease in feedlot cattle. IMPORTANCE Bovine respiratory disease (BRD) remains the most significant health challenge affecting the North American beef cattle industry and results in $3 billion in economic losses yearly. Current BRD control strategies mainly rely on antibiotics, with metaphylaxis commonly employed to mitigate BRD incidence in commercial feedlots. However, the emergence of multidrug-resistant BRD pathogens threatens to reduce the efficacy of antimicrobials. Here, we investigated the potential use of novel bacterial therapeutics (BTs) to modulate the nasopharyngeal microbiota in beef calves, which are commonly administered metaphylactic antibiotics to mitigate BRD when sourced from auction markets. By direct comparison of the BTs with an antibiotic commonly used for BRD metaphylaxis in feedlots, this study conveyed the potential use of the BTs to modulate respiratory microbiome and thereby improve resistance against BRD in feedlot cattle.

Corrigendum: Phenotypic and genetic parameters of circadian rhythms from core body temperature profiles and their relationships with beef steers' production efficiency profiles during successive winter feeding periods.

[This corrects the article DOI: 10.3389/fgene.2023.1026601.].

Phenotypic and genetic parameters of circadian rhythms from core body temperature profiles and their relationships with beef steers' production efficiency profiles during successive winter feeding periods.

This 2-year study evaluated differences in circadian parameters obtained from measures of core body temperatures using telemetric reticulo-rumen and rectal devices during two winter feeding regimes in western Canada. The study also estimated phenotypic correlations and genetic parameters associated with circadian parameters and other production traits in each feeding regime. Each year, 80 weaned steer calves (initial age: 209 ± 11 days; BW: 264 ± 20 kg) from the same cohort were tested over two successive regimes, Fall-Winter (FW) and Winter-Spring (WS) at Lanigan, Saskatchewan, Canada. The steers received forage-based rations in both regimes where the individual feed intake was measured with automatic feeding units. During the trial, the reticulo-rumen (RTMP) and rectal (RCT) temperatures were simultaneously measured every 5 min using telemetric devices. These were used to calculate the circadian parameters (Midline Estimating Statistic Of Rhythms, amplitude, and acrophase/peak time) for both temperature measures. Growth and efficiency performance traits were also determined for all steers. Each steer was assigned into inefficient, neutral, and efficient classes based on the SD of the residual feed intake (RFI), residual gain (RG), and residual intake and gain (RIG) within each year and feeding regime. Higher (p < 0.0003) RTMP and rectal temperature MESORs were observed in the Fall-Winter compared to the Winter-Spring regime. While the two test regimes were different (p < 0.05) for the majority of the RTMP or RCT temperature parameters, they did not differ (p > 0.10) with the production efficiency profiles. The heritability estimates were higher in FW (0.78 ± 0.18 vs. 0.56 ± 0.26) than WS (0.50 ± 0.18 vs. 0.47 ± 0.22) for the rumen and rectal MESORs, respectively. There were positive genetic correlations between the two regimes for the RTMP (0.69 ± 0.21) and RCT (0.32 ± 0.59). There was a negative correlation (p < 0.001) between body temperature and ambient temperature. The high heritability estimates and genetic correlations for rumen and rectal temperature parameters demonstrate their potential as beef genetic improvement tools of economic traits associated with the parameters. However, there are limited practical implications of using only the core-body temperature as a proxy for production efficiency traits for beef steers during winter.

Topography of the respiratory, oral, and guttural pouch bacterial and fungal microbiotas in horses.

BACKGROUND: The lower respiratory tract microbiota of the horse is different in states of health and disease, but the bacterial and fungal composition of the healthy respiratory tract of the horse has not been studied in detail. HYPOTHESIS: The respiratory tract environment contains distinct niche microbiotas, which decrease in species richness at more distal sampling locations. OBJECTIVE: Characterize the bacterial and fungal microbiotas along the upper and lower respiratory tract of the horse. ANIMALS: Healthy Argentinian Thoroughbred horses (n = 11) from the same client-owned herd. METHODS: Prospective cross-sectional study. Eleven upper and lower respiratory tract anatomical locations (bilateral nasal, bilateral deep nasal, nasopharynx, floor of mouth, oropharynx, arytenoids, proximal and distal trachea, guttural pouch) were sampled using a combination of swabs, protected specimen brushes, and saline washes. Total DNA was extracted from each sample and negative control, and the 16S rRNA gene (V4) and ITS2 region were sequenced. Community composition, alpha-diversity, and beta-diversity were compared among sampling locations. RESULTS: Fungal species richness and diversity were highest in the nostrils. More spatial heterogeneity was found in bacterial composition than in fungal communities. The pharyngeal microbiota was most similar to the distal tracheal bacterial and fungal microbiota in healthy horses and therefore may serve as the primary source of bacteria and fungi to the lower respiratory tract. CONCLUSIONS AND CLINICAL IMPORTANCE: The pharynx is an important location that should be targeted in respiratory microbiota research in horses. Future studies that investigate whether biomarkers of respiratory disease can be reliably detected in nasopharyngeal swab samples are warranted.

Applying multi-omics data to study the genetic background of bovine respiratory disease infection in feedlot crossbred cattle.

Bovine respiratory disease (BRD) is the most common and costly infectious disease affecting the wellbeing and productivity of beef cattle in North America. BRD is a complex disease whose development is dependent on environmental factors and host genetics. Due to the polymicrobial nature of BRD, our understanding of the genetic and molecular mechanisms underlying the disease is still limited. This knowledge would augment the development of better genetic/genomic selection strategies and more accurate diagnostic tools to reduce BRD prevalence. Therefore, this study aimed to utilize multi-omics data (genomics, transcriptomics, and metabolomics) analyses to study the genetic and molecular mechanisms of BRD infection. Blood samples of 143 cattle (80 BRD; 63 non-BRD animals) were collected for genotyping, RNA sequencing, and metabolite profiling. Firstly, a genome-wide association study (GWAS) was performed for BRD susceptibility using 207,038 SNPs. Two SNPs (Chr5:25858264 and BovineHD1800016801) were identified as associated (p-value <1 × 10-5) with BRD susceptibility. Secondly, differential gene expression between BRD and non-BRD animals was studied. At the significance threshold used (log2FC>2, logCPM>2, and FDR<0.01), 101 differentially expressed (DE) genes were identified. These DE genes significantly (p-value <0.05) enriched several immune responses related functions such as inflammatory response. Additionally, we performed expression quantitative trait loci (eQTL) analysis and identified 420 cis-eQTLs and 144 trans-eQTLs significantly (FDR <0.05) associated with the expression of DE genes. Interestingly, eQTL results indicated the most significant SNP (Chr5:25858264) identified via GWAS was a cis-eQTL for DE gene GPR84. This analysis also demonstrated that an important SNP (rs209419196) located in the promoter region of the DE gene BPI significantly influenced the expression of this gene. Finally, the abundance of 31 metabolites was significantly (FDR <0.05) different between BRD and non-BRD animals, and 17 of them showed correlations with multiple DE genes, which shed light on the interactions between immune response and metabolism. This study identified associations between genome, transcriptome, metabolome, and BRD phenotype of feedlot crossbred cattle. The findings may be useful for the development of genomic selection strategies for BRD susceptibility, and for the development of new diagnostic and therapeutic tools.

Comparison of pathogenic bacteria in the upper and lower respiratory tracts of cattle either directly transported to a feedlot or co-mingled at auction markets prior to feedlot placement.

Introduction: Bacterial bronchopneumonia (BP) has been associated with purchasing cattle through auction markets. However, whether auction markets are a source of BP-associated bacterial pathogens is unknown. This study evaluated prevalence, antimicrobial susceptibility, and genetic relatedness (using pulsed-field gel electrophoresis, PFGE) of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolated from cattle either transported to an auction market prior to feedlot placement (AUC), or directly to a feedlot from a farm (RANC). Methods: Two groups of cattle were enrolled (N = 30 per group) from two separate farms with 15 animals from an individual farm designated as AUC or RANC. Deep nasal swab (DNS) and trans-tracheal aspirates (TTA) were collected on day 0 at weaning (T0) and on day 2 at on-arrival processing at the feedlot (T1). The DNS were also collected on day 9 (T2) and day 30 (T3) after arrival at the feedlot. Results and discussion: In both TTA and DNS, prevalence of bacteria did not differ between AUC and RANC groups (P > 0.05). None of the bacteria isolated at T0 were resistant to antimicrobials and diversity of all bacteria was greatest at T0 and T1. In Group 1 cattle, 100% of P. multocida isolated at T2 and T3 were multi-drug resistant. These isolates were highly related (>90%) according to PFGE, with most being clones. Though limited in size, results for animals evaluated in this study suggested that auction markets were not a major source of resistant BP pathogens, however, horizontal transmission of a multi-resistant strain of P. multocida occurred in a feedlot. Spread of resistant P. multocida was likely due to the selective pressures imposed by feedlot antimicrobial use and encoded resistance by the bacteria.

Evaluation of reticulorumen temperature boluses for the diagnosis of subclinical cases of bovine respiratory disease in feedlot cattle.

Bovine respiratory disease (BRD) is the most important and costly health issue of the feedlot industry worldwide. Remote monitoring of reticulorumen temperature has been suggested as a potential tool to improve the diagnostic accuracy of BRD. The present study aimed to evaluate 1) the difference and degree of reticulorumen hyperthermia episodes between healthy and subclinical BRD feedlot steers, and 2) determine the correlation between reticulorumen hyperthermia and lung pathology, performance, and carcass traits. Mixed-breed feedlot steers (n = 148) with a mean arrival weight of 321 ± 3.34 kg were administered a reticulorumen bolus at feedlot entry and monitored for visual and audible signs of BRD until slaughter when lungs were examined and scored for lesions indicative of BRD. Post-slaughter animals with no record of BRD treatment were assigned to one of three case definitions. Healthy steers had no visual or audible signs of BRD (i.e., CIS=1), and total lung consolidation score < 5% or pleurisy score < 3 at slaughter. Subclinical BRD cases had a CIS of 1, and a lung consolidation score ≥ 5% or a pleurisy score of 3 at slaughter. Mild CIS cases had at least one CIS of 2, and a lung consolidation score < 5% and a pleurisy score < 3 at slaughter. Subclinical BRD and mild CIS cases had longer total duration of reticulorumen hyperthermia, more episodes and longer average episode duration above 40.0 °C compared to healthy steers (P < 0.05). A moderate positive correlation was found between lung consolidation and total duration (r = 0.27, P < 0.001), episode duration (r = 0.29, P < 0.001), and number of episodes (r = 0.20, P < 0.05). Pleurisy score was also found to be moderately and positively correlated with total duration (r = 0.23, P < 0.01), episode duration (r = 0.37, P < 0.001), and number of episodes (r = 0.26, P < 0.01). Moderate negative correlations were found between reticulorumen hyperthermia and carcass traits including hot standard carcass weight (HSCW) (-0.22 ≤ r ≤ -0.23, P < 0.05) and P8-fat depth (-0.18 ≤ r ≤ -0.32, P < 0.05). Subclinical BRD reduced carcass weight by 22 kg and average daily gain (ADG) by 0.44 kg/day compared to healthy steers (P < 0.05), but mild CIS cases had no effect on performance (P > 0.05). The reticulorumen bolus technology appears promising for detection of subclinical BRD cases in feedlot cattle as defined by lung pathology at slaughter.

Prediction of Mannheimia haemolytica serotypes based on whole genomic sequences.

The aim of the investigation was to predict the serotypes of M. haemolytica based on whole genomic sequences with the capsular gene region as target. A total of 22 strains selected to have been serotyped and to represent all serotypes were investigated by whole genomic sequencing. The BIGSdb (Bacterial Isolate Genome Sequence Database) was downloaded and installed on a Linux server. Here the sequence database was setup with unique loci at serotype level. The server allows serotypes of M. haemolytica to be predicted from whole genomic sequences and the service is available to the public for free from https://ivsmlst.sund.root.ku.dk.

Whole-Blood Transcriptome Analysis of Feedlot Cattle With and Without Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is one of the main factors leading to morbidity and mortality in feedlot operations in North America. A complex of viral and bacterial pathogens can individually or collectively establish BRD in cattle, and to date, most disease characterization studies using transcriptomic techniques examine bronchoalveolar and transtracheal fluids, lymph node, and lung tissue as well as nasopharyngeal swabs, with limited studies investigating the whole-blood transcriptome. Here, we aimed to identify differentially expressed (DE) genes involved in the host immune response to BRD using whole blood and RNA sequencing. Samples were collected from heifers (average arrival weight = 215.0 ± 5.3 kg) with (n = 25) and without (n = 18) BRD at a commercial feedlot in Western Canada. RNAseq analysis showed a distinct whole-blood transcriptome profile between BRD and non-BRD heifers. Further examination of the DE genes revealed that those involved in the host inflammatory response and infectious disease pathways were enriched in the BRD animals, while gene networks associated with metabolism and cell growth and maintenance were downregulated. Overall, the transcriptome profile derived from whole blood provided evidence that a distinct antimicrobial peptide-driven host immune response was occurring in the animals with BRD. The blood transcriptome of the BRD animals shows similarities to the transcriptome profiles obtained from lung and bronchial lymph nodes in other studies. This suggests that the blood transcriptome is a potential diagnostic tool for the identification of biomarkers of BRD infection and can be measured in live animals and used to further understand infection and disease in cattle. It may also provide a useful tool to increase the understanding of the genes involved in establishing BRD in beef cattle and be used to investigate potential therapeutic applications.

Progression of nasopharyngeal and tracheal bacterial microbiotas of feedlot cattle during development of bovine respiratory disease.

It is generally accepted that as bovine respiratory disease (BRD) develops, bacterial pathogens first proliferate in the nasopharynx and then colonize the lungs, leading to bronchopneumonia. However, such temporal changes have never been definitively demonstrated. Therefore, the objective was to describe the progression of the nasopharyngeal and tracheal bacterial microbiotas of feedlot cattle during development of BRD. Nasopharyngeal swabs and tracheal wash samples were collected from 24 heifers over 20 d after arrival at a feedlot. Heifers were assessed daily and sampled based on reticulo-rumen/rectal temperatures and development of clinical signs of BRD. The study end point for each heifer was either at BRD treatment (BRD group; n = 15) or day 20 if the heifer remained healthy or did not meet BRD treatment criteria (TOL group; n = 9). Total DNA was extracted from each sample and the 16S rRNA gene (V3-V4) sequenced. Alpha and beta diversity were compared between BRD-TOL groups and sampling locations over time. There were no common patterns of change over time in composition or diversity of either the nasopharyngeal or tracheal bacterial microbiotas of cattle that developed BRD. Health status affected bacterial composition (R2 = 0.043; < 0.001), though this effect was low compared to variation among individual animals (R2 = 0.335; < 0.001) and effects of days on feed (R2 = 0.082; < 0.001). Specific bacterial taxa (Moraxella and Mycoplasma dispar) nevertheless appeared to have a potential role in respiratory health.

Topography of the respiratory tract bacterial microbiota in cattle.

BACKGROUND: Bacterial bronchopneumonia (BP) is the leading cause of morbidity and mortality in cattle. The nasopharynx is generally accepted as the primary source of pathogenic bacteria that cause BP. However, it has recently been shown in humans that the oropharynx may act as the primary reservoir for pathogens that reach the lung. The objective was therefore to describe the bacterial microbiota present along the entire cattle respiratory tract to determine which upper respiratory tract (URT) niches may contribute the most to the composition of the lung microbiota. METHODS: Seventeen upper and lower respiratory tract locations were sampled from 15 healthy feedlot steer calves. Samples were collected using a combination of swabs, protected specimen brushes, and saline washes. DNA was extracted from each sample and the 16S rRNA gene (V3-V4) was sequenced. Community composition, alpha-diversity, and beta-diversity were compared among sampling locations. RESULTS: Microbiota composition differed across sampling locations, with physiologically and anatomically distinct locations showing different relative abundances of 1137 observed sequence variants (SVs). An analysis of similarities showed that the lung was more similar to the nasopharynx (R-statistic = 0.091) than it was to the oropharynx (R-statistic = 0.709) or any other URT sampling location. Five distinct metacommunities were identified across all samples after clustering at the genus level using Dirichlet multinomial mixtures. This included a metacommunity found primarily in the lung and nasopharynx that was dominated by Mycoplasma. Further clustering at the SV level showed a shared metacommunity between the lung and nasopharynx that was dominated by Mycoplasma dispar. Other metacommunities found in the nostrils, tonsils, and oral microbiotas were dominated by Moraxella, Fusobacterium, and Streptococcus, respectively. CONCLUSIONS: The nasopharyngeal bacterial microbiota is most similar to the lung bacterial microbiota in healthy cattle and therefore may serve as the primary source of bacteria to the lung. This finding indicates that the nasopharynx is likely the most important location that should be targeted when doing bovine respiratory microbiota research. Video abstract.

Respiratory Bacterial Microbiota in Cattle: From Development to Modulation to Enhance Respiratory Health.

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. The resulting microbiota can be diverse and enriched with known beneficial bacteria that can provide colonization resistance against bacterial pathogens or, on the contrary, with opportunistic pathogens that can predispose cattle to respiratory disease. The respiratory microbiota can be modulated by nonantimicrobial approaches to promote health, creating new potential strategies for prevention and treatment of bovine respiratory disease.

Intranasal Bacterial Therapeutics Reduce Colonization by the Respiratory Pathogen Mannheimia haemolytica in Dairy Calves.

Six Lactobacillus strains originating from the nasopharyngeal microbiota of cattle were previously characterized in vitro and identified as candidate bacterial therapeutics (BTs) for mitigating the bovine respiratory pathogen Mannheimia haemolytica In the present study, these BT strains were evaluated for their potential to (i) reduce nasal colonization by M. haemolytica, (ii) modulate the nasal microbiota, and (iii) stimulate an immune response in calves experimentally challenged with M. haemolytica. Twenty-four Holstein bull calves (1 to 3 weeks old) received either an intranasal BT cocktail containing 6 Lactobacillus strains (3 × 109 CFU per strain; BT + Mh group) 24 h prior to intranasal M. haemolytica challenge (3 × 108 CFU) or no BTs prior to challenge (Mh, control group). Nasal swab, blood, and transtracheal aspiration samples were collected over the course of 16 days after BT inoculation. Counts of M. haemolytica were determined by culturing, and the nasal and tracheal microbiotas were evaluated using 16S rRNA gene sequencing. Serum cytokines (interleukin-6 [IL-6], IL-8, and IL-10) were quantified by enzyme-linked immunosorbent assay (ELISA). Administration of BT reduced nasal colonization by M. haemolytica (P = 0.02), modified the composition and diversity of the nasal microbiota, and altered interbacterial relationships among the 10 most relatively abundant genera. The BT + Mh calves also had a lower relative abundance of Mannheimia in the trachea (P < 0.01) but similar cytokine levels as Mh calves. This study demonstrated that intranasal BTs developed from the bovine nasopharyngeal Lactobacillus spp. were effective in reducing nasal colonization by M. haemolytica in dairy calves.IMPORTANCE Bovine respiratory disease (BRD) is one of the significant challenges for the modern dairy industry in North America, accounting for 23 to 47% of the total mortality among pre- and postweaned dairy heifers. Mass medication with antibiotics is a common practice to control BRD in dairy cattle. However, the emergence of multidrug-resistant BRD pathogens highlights the importance of developing alternatives to antibiotics for BRD mitigation. Using a targeted approach, we recently identified 6 Lactobacillus strains originating from the bovine respiratory microbiota as candidates to be used as bacterial therapeutics (BTs) for the mitigation of the BRD pathogen Mannheimia haemolytica Here, we demonstrated that intranasal inoculation of the BT strains reduced nasal colonization by M. haemolytica in dairy calves experimentally challenged with this pathogen. This study, for the first time, shows the potential use of intranasal BTs as an alternative to mitigate BRD pathogens in cattle.

Genetic relatedness and antimicrobial resistance in respiratory bacteria from beef calves sampled from spring processing to 40 days after feedlot entry.

Recent studies have shown an increase in antimicrobial-resistant bovine respiratory disease (BRD) pathogens. To investigate the origin of antimicrobial resistance in the respiratory microbiota of beef cattle, three groups (A, B, or C) of 40 calves sourced from different calf-ranches were sampled by deep nasopharyngeal swab (DNS) at the time of first on-ranch vaccination (Time point 1, T1), feedlot entry (Time point 2, T2), and 40 days after feedlot entry (Time point 3, T3; feedlots differed by group). Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni were isolated from DNS samples, tested for antimicrobial susceptibility, and subtyped by pulsed-field gel electrophoresis (PFGE). Antimicrobial resistance genes [tet(H), tet(W), and sul2] were also quantified in DNS metagenomic DNA using PCR. Prevalence of calves positive for BRD pathogens differed among groups and time-points but P. multocida was the most prevalent (61% of calves positive, at least, at one timepoint), followed by M. haemolytica (48%) and H. somni (26%). Most M. haemolytica were susceptible to all antimicrobials (88.6%; n = 70). For P. multocida, the dominant resistance phenotype was against oxytetracycline and neomycin (35.8%). Resistant P. multocida isolates were mainly detected in group C at T3 and had the same PFGE profile. For H. somni, the dominant resistance phenotype was against neomycin (63.3%) and was only observed at T3. The abundance of tet(W) did not change significantly over time (P > 0.05). Abundances of tet(H) and sul2 only increased for group C at T3 (P < 0.05). Overall, this study showed that resistance in the respiratory microbiota of beef calves can increase from calf-ranch to feedlot however, the results can vary by calf-ranch and feedlot.

The role of the bovine respiratory bacterial microbiota in health and disease.

Increased antimicrobial resistance in bovine respiratory bacterial pathogens poses a threat to the effective control and prevention of bovine respiratory disease (BRD). As part of continued efforts to develop antimicrobial alternatives to mitigate BRD, the microbial community residing within the respiratory tract of feedlot cattle has been increasingly studied using next-generation sequencing technologies. The mucosal surfaces of upper and lower respiratory tracts of cattle are colonized by a diverse and dynamic microbiota encompassing commensal, symbiotic, and pathogenic bacteria. While a direct causal relationship between respiratory microbiota and the development of BRD in feedlot cattle has not been fully elucidated, increasing evidence suggests that the microbiota contributes to respiratory health by providing colonization resistance against pathogens and maintaining homeostasis. Certain management practices such as weaning, transportation, feed transition, and antibiotic application can disrupt the respiratory microbiota, potentially altering pathogen colonization. Microbiota-based approaches, including bacterial therapeutics that target restoring the normal respiratory microbiota, may provide new methods for mitigating BRD in feedlot cattle in place of antibiotics. In addition, the distinct bacterial respiratory microbial communities observed in BRD-affected and healthy feedlot cattle may allow for future application of microbiota-based techniques used in the diagnosis of BRD.

Evaluation of the Nasopharyngeal Microbiota in Beef Cattle Transported to a Feedlot, With a Focus on Lactic Acid-Producing Bacteria.

The nasopharyngeal (NP) microbiota is important in defining respiratory health in feedlot cattle, with certain NP commensals potentially protecting against bovine respiratory disease (BRD) pathogens. In the present study, we evaluated longitudinal changes in the NP microbiota with a focus on lactic acid-producing bacteria (LAB) and their linkage with BRD-associated bacteria in steers (n = 13) that were first transported to an auction market, and then to a feedlot. Deep nasopharyngeal swabs were collected at the farm before transportation to the auction market (day 0), at feedlot placement (day 2), and 5 (day 7) and 12 (day 14) days after feedlot placement. Swabs were processed for the assessment of the NP microbiota using 16S rRNA gene sequencing, and for the detection of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni by culturing. Possible associations among the top 15 most relatively abundant bacterial genera were predicted using a stepwise-selected generalized linear mixed model. Correlations between LAB and BRD-associated Pasteurellaceae families were also assessed. In addition, antimicrobial activity of selected LAB isolates against M. haemolytica was evaluated in vitro. A noticeable shift was observed in the NP microbial community structure, and in the relative abundance of LAB families as a result of auction market exposure, transport and feedlot placement. Varying degrees of positive or negative associations between the 15 most relatively abundant genera were observed. Many of the LAB families were inversely correlated with the BRD-associated Pasteurellaceae family as the cattle were transported to the auction market and then to the feedlot. Nearly all steers were culture-negative for M. haemolytica and H. somni, and P. multocida became less prevalent after feedlot placement. Isolates from the Lactobacillaceae, Streptococcaceae, and Enterococcaceae families inhibited the growth of M. haemolytica. The results of this study indicated that the NP microbiota became more diverse with an increase in microbial richness following transport to an auction market and feedlot. This study provides evidence of potential cooperation and exclusion taking place in the respiratory microbial community of cattle which may be useful for developing microbial-based strategies to mitigate BRD.

Development of Bacterial Therapeutics against the Bovine Respiratory Pathogen Mannheimia haemolytica.

Bovine respiratory disease (BRD) is a major cause of morbidity and mortality in beef cattle. Recent evidence suggests that commensal bacteria of the bovine nasopharynx have an important role in maintaining respiratory health by providing colonization resistance against pathogens. The objective of this study was to screen and select bacterial therapeutic candidates from the nasopharynxes of feedlot cattle to mitigate the BRD pathogen Mannheimia haemolytica In a stepwise approach, bacteria (n = 300) isolated from the nasopharynxes of 100 healthy feedlot cattle were identified and initially screened (n = 178 isolates from 12 different genera) for growth inhibition of M. haemolytica Subsequently, selected isolates were evaluated for the ability 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 of M. haemolytica, with 88% of the isolates (n =33) having inhibition zones ranging from 17 to 23 mm. Adherence to BT cells ranged from 3.4 to 8.0 log10 CFU per 105 BT cells. All the isolates tested in competition assays reduced M. haemolytica adherence to BT cells (32% to 78%). Among 84 bovine genes evaluated, selected isolates upregulated expression of interleukin 8 (IL-8) and IL-6 (P < 0.05). After ranking isolates for greatest inhibition, adhesion, competition, and immunomodulation properties, 6 Lactobacillus strains from 4 different species were selected as the best candidates for further development as intranasal bacterial therapeutics to mitigate M. haemolytica infection in feedlot cattle.IMPORTANCE Bovine respiratory disease (BRD) is a significant animal health issue impacting the beef industry. Current BRD prevention strategies rely mainly on metaphylactic use of antimicrobials when cattle enter feedlots. However, a recent increase in BRD-associated bacterial pathogens that are resistant to metaphylactic antimicrobials highlights a pressing need for the development of novel mitigation strategies. Based upon previous research showing the importance of respiratory commensal bacteria in protecting against bronchopneumonia, this study aimed to develop bacterial therapeutics that could be used to mitigate the BRD pathogen Mannheimia haemolytica Bacteria isolated from the respiratory tracts of healthy cattle were characterized for their inhibitory, adhesive, and immunomodulatory properties. In total, 6 strains were identified as having the best properties for use as intranasal therapeutics to inhibit M. haemolytica If successful in vivo, these strains offer an alternative to metaphylactic antimicrobial use in feedlot cattle for mitigating BRD.

Draft Genome Sequences of 14 Lactobacillus, Enterococcus, and Staphylococcus Isolates from the Nasopharynx of Healthy Feedlot Cattle.

Here, we present the first draft genome sequences of 14 bacterial strains isolated from the nasopharynx of healthy feedlot cattle. These genomes are from 12 Lactobacillus isolates (L. amylovorus, L. buchneri, L. curvatus, and L. paracasei), 1 Enterococcus hirae isolate, and 1 Staphylococcus chromogenes isolate.

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.

The role of the respiratory bacterial microbiota in the pathogenesis of bovine respiratory disease (BRD) is still not well defined, limiting our understanding of the disease. Specifically, there is no information on the nasopharyngeal bacterial microbiota of cattle raised without antimicrobials. The objective was to characterize and compare the nasopharyngeal bacterial microbiota in feedlot cattle raised without antimicrobials that were healthy or diagnosed with BRD. Newly-received feedlot cattle (arrival bodyweight ± SD = 218 ± 37 kg) with BRD (n = 82) and pen-matched controls (n = 82) were clinically examined and sampled by deep nasopharyngeal swab (DNS). DNA was extracted from each DNS and the 16S rRNA gene (V4) was sequenced. Alpha and beta diversity were compared between health groups and among 3 days-on-feed (DOF) groups (group A = 3-12 DOF; group B = 13-20 DOF; group C = 21-44 DOF). Observed species richness was lower (P = 0.031) in cattle with BRD compared to healthy ones. Both health status (P = 0.007) and DOF groups (P < 0.001) were sources of variation in microbiota composition. Differences between health groups were driven by multiple sequence variants, including Mycoplasma bovis, Histophilus somni, and several Moraxella spp. Notably, M. bovis was more frequently identified in cattle with BRD. M. bovis identification was also higher in cattle sampled at later DOF. The increased identification of M. bovis in cattle with BRD reaffirms a potentially significant role for this bacterium in respiratory health.