Mannheimia haemolytica is a pathobiont bacterium of the upper respiratory tract in cattle and is associated with acute bovine respiratory disease (BRD). Despite research into bacterial pathogens and their associations with BRD, little is known about how bacterial variant dynamics interplay with disease progression. Therefore, our objective was to explore the clonal diversity of M. haemolytica in beef cattle utilizing whole genome sequencing. Ninety-nine M. haemolytica isolates from a commingled cohort of 33 cattle sampled in a prior study were selected for analysis. Long-read whole-genome sequencing was conducted to evaluate antimicrobial resistance genes, virulence factors, plasmids, and single nucleotide polymorphisms. Clonal lineages were derived using multi-locus sequence typing (MLST), a recently developed genomic sequence variant (GSV) method, and a core SNP-based clustering analysis with the average linkage method. We identified no differences in pathogenic profiles across isolates. We identified a single sequence type (ST1) and a single GSV (GSV 2) utilizing the MLST and GSV methods, respectively. The core SNP-based average linkage clustering analysis produced different clonal lineage estimations depending on the core SNP cutoff values (2 to 18 clusters). All estimations were greater than the single sequence type and GSV suggested by the MLST and the GSV methods. BRD status did not correlate with clonal interpretations. Our results suggest that method selection can influence the clonal interpretation of M. haemolytica within a population but may not reflect pathogenic capability. Alternate methods that increase discriminatory power or transcriptional profiles may provide new insights and should be evaluated.IMPORTANCEBovine respiratory disease remains a major challenge for cattle health and production, yet the population dynamics of key bacterial pathogens remain poorly understood. Using long-read whole-genome sequencing, this study shows that clonal diversity estimates of Mannheimia haemolytica are highly dependent on the analytical method employed, despite uniform virulence gene content. These findings highlight the importance of method selection in genomic epidemiology and caution against overinterpreting clonal structure as a proxy for pathogenic potential.

Objective:To utilize full-length 16S rRNA long-read sequencing to (1) describe the microbiome profile of synovial fluid, previously classified by cytology and microbial culture, from foals with and without septic arthritis and (2) determine the agreement between the microbiome profiles and microbial culture. Methods:A retrospective observational study was conducted using synovial samples collected from foals treated at a single equine referral center (from 2018 through 2024). The synovial fluid was classified as septic or not septic using standard cytological and microbial culture methods. Deoxyribonucleic acid was extracted from stored samples and sequenced using a MinION sequencer. Linear mixed modeling was used to determine the relationships between α diversity and age, affected joint, and cytological and culture status. β diversity was assessed using permutational multivariate ANOVA. Differential taxa were identified with ALDEx2. The agreement between sequencing and culture was evaluated using the Gwet coefficient. Results:Fifty samples met inclusion criteria. α diversity (Shannon index) was higher in culture-positive samples (mean difference, 0.42; 95% CI, 0.12, 0.72). Microbiome composition differed by culture (R2 for Aitchison dissimilarity distance, 0.091) and cytology status (R2 for Jaccard dissimilarity distance, 0.029). No taxa differed by age, joint, or cytology. Klebsiella were significantly more abundant in culture-positive samples (ALDEx2 standardized effect size, 0.65; 95% CI, -1.63, 7.01). Agreement between positive culture and the predominant sequenced genus was 0.774 (Gwet AC1 coefficient) with a probabilistic benchmark interval of 0.6 to 0.8 (substantial agreement). Conclusions:Next-generation sequencing and culture results were substantially aligned. Clinical Relevance:Next-generation sequencing may aid diagnosis in suspected septic arthritis cases with negative culture results.

Mycoplasma bovis is a significant bacterial pathogen involved in the Bovine Respiratory Disease (BRD) complex. While assessing localized mucosal immunity is vital for understanding host defenses against respiratory pathogens, there is a lack of validated diagnostic assays for detecting mucosal immunoglobulins. This study aimed to evaluate the diagnostic performance of a novel multiplex ELISA platform (Pictor PictVet®) in detecting anti-M. bovis IgG in bovine nasopharyngeal swab washes. Archived nasopharyngeal swab washes from crossbred feedlot steers (50 positive and 50 negative for M. bovis DNA via digital qPCR) were evaluated. The ELISA utilized three target antigens: K-0310, K-0320, and MilA. Diagnostic accuracy was assessed using both concurrent M. bovis DNA status and a Latent Class Analysis (LCA) model as ground truths. Inter-operator reproducibility and analytical sensitivity (by serial dilution) were also determined. The MilA antigen exhibited high variance and poor predictive performance (51.5% accuracy), resulting in its exclusion from the final models. When utilizing M. bovis DNA status as the ground truth, the best custom cutoff (K-0310 and K-0320 in series) yielded high specificity (88%) but low sensitivity (30%). However, employing LCA to estimate IgG status (17.1% prevalence) dramatically improved diagnostic validity. Evaluating K-0310 and K-0320 in parallel achieved an accuracy of 97.59% with 100% sensitivity and 95.18% specificity. The assay demonstrated excellent inter-operator agreement (Kappa = 0.894 to 1) and reliably detected positive samples at a 1:2 dilution. Analyzing nasopharyngeal swabs with this novel multiplex ELISA platform, specifically utilizing the K-0310 and K-0320 antigens, provides a highly accurate, reproducible, and non-invasive method for monitoring mucosal anti-M. bovis IgG. The use of LCA was paramount in establishing appropriate cutoffs, highlighting the limitations of relying solely on pathogen DNA detection to evaluate adaptive immune responses.

Strain variation plays a key role in the microbial epidemiology of Mycoplasma bovis, yet its true diversity remains incompletely characterized, partly due to limitations of culture-based methods. This study evaluated the in silico suitability of a targeted enrichment (TE) shotgun sequencing approach to detect and classify M. bovis strains in milk metagenomic samples. As a proof of concept, the accuracy of this approach was assessed using milk-derived M. bovis strains. A total of 620 M. bovis whole-genome sequences were downloaded from NCBI, of which 162 (26.1%) originated from milk samples. Genomes were grouped into Genomically Clustered Sequence Variants (GSVs) using MashTree and TreeCluster to enable strain-level classification. To simulate TE sequencing data, genomes from different milk-associated GSVs were randomly selected and fragmented in silico into 150-bp reads. Mock milk samples were generated by sampling reads with replacement from these genomes. Sequencing depth was modeled using a Poisson distribution, while mixed-strain DNA samples were simulated by including 1, 3, 6, or 9 GSVs per sample. Enrichment proportions were set at 0.3, 0.5, 0.7, and 0.9. Two classification tools, Kraken2 and Themisto/mSWEEP, were evaluated for their ability to detect and classify the simulated TE reads. Themisto/mSWEEP consistently outperformed Kraken2, achieving an average read classification accuracy of 84.9% compared with 1.4% for Kraken2. Sensitivity for Themisto/mSWEEP was 100% with a single spiked GSV and declined slightly to 97.0% with nine GSVs, whereas Kraken2 achieved sensitivities of only 17.3% and 4.7%, respectively. Positive predictive value (PPV) showed a similar pattern: 98% for Themisto/mSWEEP vs. 4.7% for Kraken2 with a single GSV, and 65.5% vs. 10% with nine GSVs. While Kraken2's PPV increased slightly with additional GSVs, Themisto/mSWEEP's PPV decreased. Both methods maintained high specificity and negative predictive value (>91%) across all scenarios. Enrichment proportion had no measurable effect on performance. Overall, Themisto/mSWEEP demonstrated superior accuracy for GSV-level identification of M. bovis strains. Enrichment to at least 30% of total reads was sufficient to recover strain-level data. Further work is needed to assess the biological relevance and practical applications of these genomic clusters.

Pathogens can shape their host communities over various timescales. The potential role of host–pathogen co-evolution in driving contemporary shifts in disease ecology is becoming increasingly important as host species emerge and persist outside their native ranges. In North America, Mycoplasma ovipneumoniae can cause fatal pneumonia epizootics in native bighorn sheep (Ovis canadensis), whereas introduced free-ranging sympatric aoudad (Ammotragus lervia) typically act as asymptomatic reservoirs. We describe the responses of two hosts with different biogeographical histories to shared pathogen exposure through a lens of potential host–pathogen co-evolution. Specifically, this work integrates findings on microbiome composition and host transcriptomic responses in aoudad and bighorn sheep before and after controlled exposure to M. ovipneumoniae, with or without leukotoxigenic Pasteurellaceae. Aoudad maintained significantly higher microbial richness (Shannon) in the lower respiratory tract, whereas bighorn sheep experienced post-exposure microbiome perturbations and enhanced growth of some opportunistic taxa. Both molecular detection of and humoral antibodies to M. ovipneumoniae reduced the relative abundance of key genera (e.g., Bibersteinia, Mannheimia, Pasteurella, Roseomonas) in the upper respiratory tract, but post-exposure bacterial community alteration was more pronounced in bighorn sheep. Transcriptome profiling revealed that, compared to aoudad, bighorn sheep upregulated pro-inflammatory and oxidative-stress pathways—including interleukin-1, interleukin-12, and NF-κB signaling—alongside reactive oxygen species generation. In contrast, aoudad exhibited comparatively muted inflammatory signatures, enhanced expression of molecular chaperones, antigen-processing machinery, and integrin-mediated regulatory genes (notably CD46, ILK, and NFKBIZ). Network analysis identified distinct hub genes likely underpinning effective pathogen clearance and mucosal resilience in aoudad versus immunopathology in bighorn sheep. Our integrated microbiome and transcriptomic data underscore the importance of co-evolutionary history in driving host-specific responses to shared respiratory pathogens. Aoudad display microbiome stability and balanced immunoregulation, whereas bighorn sheep suffer dysbiosis and excessive inflammation, potentially increasing mortality risk. Explicit consideration of evolutionary and ecological context regarding host–pathogen co-evolution may increase overall understanding of observed pathobiological and epidemiological patterns commonly targeted for disease management interventions.

High-resolution genomic characterization is essential for understanding diversity, pathogenicity, and transmission dynamics of bacterial pathogens. Mannheimia haemolytica (Mh) is the most consequential bacterial agent associated with bovine respiratory disease (BRD) in cattle, as a leading cause of morbidity, mortality, and antimicrobial use. Historically, BRD pathogens, including Mh, have been studied using culture or PCR approaches that provided limited ability to characterize fine-scale genomic variation across communities. Here, we evaluated target-enriched (TE) shotgun sequencing, a culture-independent method capable of strain-level resolution within metagenomic data, for detecting and characterizing Mh in comparison with qPCR and 16S rRNA gene sequencing. Nasal swabs (10 individual and 2 composited DNA samples per pen) and environmental samples (three ropes hung on pen rails and three water bowl swabs per pen) were collected from four pens in each of five distinct cattle populations. DNA was extracted for TE sequencing to identify Mh at both species and genomic sequence variant (GSV) levels, and to characterize antimicrobial resistance genes across the bacterial communities. qPCR was performed to quantify Mh genome copies, and 16S rRNA gene sequencing was used to assess the broader respiratory microbiome. TE sequencing identified Mh in 100% of TE-tested samples and classified multiple GSVs in all but 3 of 121 samples. GSV profiles clustered within housing groups and varied across cattle populations, indicating structured strain-level diversity. In contrast, Mannheimia spp. were detected in only 47.7% of samples by 16S rRNA sequencing. These findings demonstrate that TE sequencing enables sensitive, strain-level characterization of Mh in cattle and environmental samples and reveals substantial within-population genomic diversity not captured by conventional approaches.IMPORTANCETarget-enriched shotgun sequencing enabled sensitive, strain-level detection of Mannheimia haemolytica (Mh), revealing multiple co-circulating genomic sequence variants (GSVs) within and among cattle groups. This demonstrates greater genetic variability of Mh populations in beef cattle than has been previously recognized. The clustering of GSVs within housing groups, together with the overlap between respiratory and environmental samples, is consistent with the hypothesis that contagious transmission contributes to Mh ecology. These results highlight the potential utility of composite nasal swab and environmental samples for future studies evaluating relationships between Mh genomic variation and disease risk.

Characterizing microbial genetic sequences and key variants is critical for understanding pathogen ecology, transmission, and clinical impact. Yet, conventional metagenomic sequencing often yields too few on-target reads to move beyond species-level identification. We developed a target-enriched (TE) metagenomic workflow, including bait design, an optimized TE shotgun protocol, and the VARIANT++ pipeline, to recover and classify reads at a clustered genomic sequence-variant (GSV) level (see Graphical abstract). The computational component clusters reference genomes by average nucleotide identity, builds a GSV database, and integrates Kraken2, Themisto, and mSWEEP to increase call confidence while reducing false positives. Using Mannheimia haemolytica ( Mh ), the primary cause of bovine respiratory disease, we designed 114,375 DNA baits targeting sequences across 70 reference genomes. TE libraries from nasopharyngeal swabs of feedlot cattle achieved >250-fold increases in on-target Mh reads (∼2.5% of non-host reads on average) compared with conventional shotgun sequencing, despite using one-quarter the sequencing depth. This variant-level resolution revealed six GSVs; most samples contained at least two, indicating variant mixtures difficult to detect with culture- or shotgun-based surveys. Because the approach leverages available reference sequences, it can be reconfigured for other microbial targets. TE metagenomics paired with genome-similarity clustering provides a scalable approach to variant-level characterization from complex microbial populations. ![Graphical abstract][1] Graphical abstract Overview of the components in our three-part workflow. ### Competing Interest Statement The authors have declared no competing interest. All sequence reads are available through BioProject PRJNA1309097 at the NCBI’s Sequence Read Archive. The code and instructions for the bioinformatic and statistical analyses can be found at the GitHub repository ([https://github.com/Microbial-Ecology-Group/Manuscript-Mh\_TE\_validation][2]) and by the corresponding DOI: 10.5281/zenodo.16989755. Texas A&M University [1]: pending:yes [2]: https://github.com/Microbial-Ecology-Group/Manuscript-Mh_TE_validation

Mycoplasma hyosynoviae (M. hyosynoviae) is a commensal bacterium that can induce lameness in growing and finishing pigs, contributing to welfare concerns in swine production. The objective of this study was to characterize the natural humoral immune response to M. hyosynoviae in various ages of pigs commercially raised in the United States. Three > 2500 sow farms and their downstream sites were enrolled in the study. One herd presented a history of M. hyosynoviae-associated lameness in growing/finishing age pigs (affected), while two farms and their downstream flows were apparently healthy (non-affected). Blood samples (n = 3395) were collected cross-sectionally at various ages and stages of production in sows, piglets, and replacement gilts. An indirect ELISA assay was employed for the detection of M. hyosynoviae IgG antibodies in serum. A Kruskal-Wallis test was utilized to investigate the differences in M. hyosynoviae S/P ratios among sows, piglets, and gilts in each herd. A logistic regression analysis was performed to assess the proportion of pigs positive for M. hyosynoviae antibodies within each herd. Sows showed the highest detection of M. hyosynoviae antibodies when compared to piglets and replacement gilts, regardless of production stage. Mycoplasma hyosynoviae antibodies tended to wane at approximately eight weeks of age in the piglets in all herds. Replacement gilt seroconversion to M. hyosynoviae in the two non-affected herds increased later in life compared to the affected flow. The lack of identification of M. hyosynoviae antibodies in the herd experiencing lameness downstream compared to the two non-affected herds warrants investigation into the role that antibody production could play against lameness development. In conclusion, this study provides information on potential windows of opportunity to induce an immune response against M. hyosynoviae.

Bovine respiratory disease (BRD) remains a priority topic with­in livestock research. A leading topic of BRD-related research is vaccination of pre-weaned calves to mitigate the effects of BRD post-weaning. Comparison of gene expression pathways in vaccinated and unvaccinated calves that develop or resist BRD would help clarify factors that may result in benefits following vaccination.

Bovine respiratory disease (BRD) is a multifactorial disease complex resulting from the interaction of various pathologic, immune-mediated and management-driven factors. Specifi­cally, management decisions around vaccination against viral components of BRD, marketing through commercial sale sys­tems, and long-haul shipping of cattle modulate the bovine im­mune system and susceptibility to disease. Therefore, we inves­tigated these factors and their influence on immunomodulation via RNA sequencing methodology.

Although bovine respiratory disease (BRD) is frequent and cost­ly in beef cattle operations, diagnosis of the disease complex is inaccurate. Metaphylactic use of antimicrobials effectively re­duces the risk of BRD, but antimicrobial administration may in­duce resistance. Therefore, this project was designed to identify differences in levels of transcribed gene expression driven by clinical BRD, both with and without administration of metaphy­laxis at-arrival in high-risk stocker cattle. Using RNA sequenc­ing, we aimed to identify predictive gene expression pathways at-arrival in cattle that developed BRD or not, characterize host genomic patterns in response to clinical BRD, and describe dif­ferences in gene expression of clinically diseased cattle based on treatment success and time of treatment within the study.

The nasal capillarid Eucoleus boehmi has gained importance in the veterinary field in recent years. However, much is still unknown regarding its life cycle, geographic distribution, and treatment. This study aimed to evaluate the efficacy of three commercial anthelmintic products against E. boehmi in foxhound dogs and to assess the olfactory capacity of infected individuals. The treatment protocols included moxidectin/imidacloprid (MI) and pyrantel pamoate/febantel/praziquantel (PFP), separately and combined, as well as emodepside/praziquantel (EP) alone. Fecal egg count reduction tests (FECRT) were performed using the mini-FLOTAC technique for evaluating the anthelmintic efficacy. The olfactory capacity was assessed through a natural detection task method. In the first phase, the fecal egg count reduction (FECR) values at day 11 post-treatment (PT) were 58 % (95 % CI: 31 %-77 %) for MI, 35 % (95 % CI: 16 %-65 %) for PFP, and 84 % (95 % CI: 68 %-92 %) for EP. On day 33 PT, the fecal egg count (FEC) was higher in all animals compared with day 0. One month after finishing the first phase of the study, a second phase of the trial was performed, and eligible animals were treated either with a combination of MI/PFP or EP alone. On day 13 PT, FECR was 95 % (95 % CI: 78 %-100 %) in animals treated with MI/PFP and 89 % (95 % CI: 77 %-94 %) in animals that received EP. While EP demonstrated moderate efficacy in both phases, MI and PFP products were ineffective individually but achieved high efficacy when used together. In the olfactory function assessment, there was a moderate negative correlation between the egg counts and the performance of the dog in the test. In general, lower FEC were associated with better performance in the olfactory assessment. Further studies are needed to evaluate additional anthelmintics' efficacy, and to further understand the effect of E. boehmi on a dog's olfactory capacity.

Bovine respiratory disease (BRD) is a major economic and animal welfare issue in the beef industry. Mycoplasma bovis is one of the main causal organisms, particularly in chronic cases. Due to the difficulty of isolating M. bovis from clinical isolates, there is a lack of information on the genetic diversity of this pathogen in the Texas panhandle region of the United States. Therefore, our objective was to provide genome-level characterization of M. bovis isolated from the lung lesions of beef and dairy cattle in the Texas panhandle. Fifty-four isolates displaying mycoplasma-like growth were recovered from bovine lung lesions by the Texas Veterinary Medical Diagnostic Laboratory in 2021 and 2022. Of these isolates, 32 were determined to be M. bovis via species-specific qPCR using the uvrC gene. Long-read whole-genome sequencing was used to identify key virulence factors, antimicrobial resistance genes, and to assess the genetic diversity of these isolates. Fisher’s exact tests were used to identify associations between isolate characteristics and host metadata, including the state of origin, type of operation, animal age, and animal sex. Our results indicate that there is considerable genetic diversity among the M. bovis isolates, despite their shared geography in the Texas panhandle, though significant clustering based on host metadata was observed. Analysis of the pangenome showed that the M. bovis isolates in this study also harbor a diverse array of virulence genes, but no antimicrobial resistance genes were identified in this study.

Abstract Cranioventral pulmonary consolidation (CVPC) is a common lesion observed in the lungs of slaughtered pigs, often associated with Mycoplasma (M.) hyopneumoniae infection. There is a need to implement simple, fast, and valid CVPC scoring methods. Therefore, this study aimed to compare CVPC scores provided by a computer vision system (CVS; AI DIAGNOS) from lung images obtained at slaughter, with scores assigned by human evaluators. In addition, intra- and inter-evaluator variability were assessed and compared to intra-CVS variability. A total of 1050 dorsal view images of swine lungs were analyzed. Total lung lesion score, lesion score per lung lobe, and percentage of affected lung area were employed as outcomes for the evaluation. The CVS showed moderate accuracy (62–71%) in discriminating between non-lesioned and lesioned lung lobes in all but the diaphragmatic lobes. A low multiclass classification accuracy at the lung lobe level (24–36%) was observed. A moderate to high inter-evaluator variability was noticed depending on the lung lobe, as shown by the intraclass correlation coefficient (ICC: 0.29–0.6). The intra-evaluator variability was low and similar among the different outcomes and lung lobes, although the observed ICC slightly differed among evaluators. In contrast, the CVS scoring was identical per lobe per image. The results of this study suggest that the CVS AI DIAGNOS could be used as an alternative to the manual scoring of CVPC during slaughter inspections due to its accuracy in binary classification and its perfect consistency in the scoring.

Abstract There is a critical lack of information on the timing of colonization of bovine respiratory disease-related bacteria in the upper respiratory tract, potentially affecting calf health and disease onset. The objective of this study was to evaluate the upper respiratory tract and fecal microbiomes of neonatal beef calves during the first 24 h of life compared with their dams at the time of birth. Late-gestation commercial beef cows (n = 28) were assigned to individual, soil-surfaced pens (6.25 x 2.29 m) fitted with canvas tarps on shared fences to limit contact between unrelated pairs. Left nasal (LN), right nasal (RN), fecal, and vaginal swab samples were collected from cows at a single time point promptly after parturition. Swabs of the LN, RN, and rectum were collected from calves at birth (0 h), 6, 12, and 24 h post-parturition. Samples were flash-frozen and stored at -80°C until processing. DNA was extracted from swabs using Power Soil Pro kit. Extracted DNA was used to prepare DNA libraries for full-length 16S rRNA gene sequencing on a MinION Mk1C device. Sequenced reads were classified using Centrifuge and used in microbiome analyses conducted in R to estimate the changes in the microbiome according to animal type (cow versus calf), time of collection, and sample type (nasal, fecal, or vaginal). Upper respiratory microbiomes significantly differed (P < 0.005) between cows and their offspring at birth. In nasal microbiomes, animal type explained 12.6% (P = 0.002) and 18.5% (P = 0.001) of variance in beta diversity in left and right cavities, respectively. Animal type explained 42.1% of the beta diversity variance in fecal microbiomes (P = 0.001). Nasal microbiomes at birth were significantly more dispersed in dams compared with their calves in both the LN (P = 0.03) and RN (P = 0.03). Similarly, fecal microbiomes of dams were significantly more dispersed (P < 0.0001) than those of their offspring. There was a clear transition of the microbial communities associated with calf age in the upper respiratory and fecal niches. Calf age accounted for 2% (P = 0.02), 3.5% (P = 0.001), and 28% (P = 0.001) of the variance in the beta diversity of the LN, RN, and fecal microbiomes, respectively. After adjusting for calf age, the laterality of nasal microbiomes was not significantly (P = 0.682) associated with the composition of microbial communities. In conclusion, calf microbial communities were dissimilar to that of their dam at birth. Additionally, microbial community transition was evident with each subsequent sampling time point, potentially influenced by the presence of the dam.

IntroductionThe gastrointestinal microbiota profoundly influences the health and productivity of animals. This study aimed to characterize microbial community structures of the mouth, gastrointestinal tract (GIT), and feces of cattle.MethodsSamples were collected from 18 Akaushi crossbred steers at harvest from multiple locations, including the oral cavity, rumen, abomasum, duodenum, jejunum, ileum, cecum, spiral colon, distal colon, and feces. These cattle were raised without exposure to antimicrobial drugs or hormone implants. Total microbial abundance was assessed using qPCR targeting the V3–V4 region of the 16S rRNA gene, and microbial community composition was evaluated through 16S rRNA gene sequencing.ResultsTotal microbial abundance was lesser in the small intestine than in other GIT regions (p ≤ 0.05). Additionally, microbial communities in the small intestine had lower richness and diversity than other regions (p ≤ 0.05). Microbial community compositions were measurably different along the GIT, with greater relatedness in adjacent GIT sections when progressing from oral to aboral locations. Firmicutes, Bacteroidota, and Actinobacteria were the dominant phyla in all samples. However, variations in composition were evident at lower taxonomic levels within these dominant phyla among samples from different regions. Genera previously associated with healthy gut microbiome communities were observed in low abundance across GIT regions. Taxa historically associated with liver abscesses (e.g., Fusobacterium and Trueperella) were detected in low abundance (≤0.02% relative abundance) throughout the GIT. In contrast, Bacteroides, which recently has been identified as a dominant feature in many liver abscesses, was observed in greater relative abundance (5.2% on average) in the hindgut.DiscussionThis study provides an in-depth evaluation of the GIT of harvest-ready Akaushi crossbred cattle of varying growth rates. Clear differences exist in the abundance and composition of microbial populations at different points of the GIT. Unfortunately, no single GIT location can adequately represent the microbial communities of the entire GIT, which has important implications for future research. Additionally, examining microbiome data only at the phylum level likely oversimplifies important complexities of the microbial community structures, and investigations of lower taxonomic ranks should be included.

Rising antimicrobial resistance (AMR) in Salmonella serotypes host-adapted to cattle is of increasing concern to the beef and dairy industry. The bulk of the existing literature focuses on AMR post-slaughter. In comparison, the understanding of AMR in Salmonella among pre-harvest cattle is still limited, particularly in Texas, which ranks top five in beef and dairy exports in the United States; inherently, the health of Texas cattle has nationwide implications for the health of the United States beef and dairy industry. In this study, long-read whole genome sequencing and bioinformatic methods were utilized to analyze antimicrobial resistance genes (ARGs) in 98 isolates from beef and dairy cattle in the Texas Panhandle. Fisher exact tests and elastic net models accounting for population structure were used to infer associations between genomic ARG profiles and antimicrobial phenotypic profiles and metadata. Gene mapping was also performed to assess the role of mobile genetic elements in harboring ARGs. Antimicrobial resistance genes were found to be statistically different between the type of cattle operation and Salmonella serotypes. Beef operations were statistically significantly associated with more ARGs compared to dairy operations. Salmonella Heidelberg, followed by Salmonella Dublin isolates, were associated with the most ARGs. Additionally, specific classes of ARGs were only present within mobile genetic elements.

Bovine respiratory disease (BRD) remains the leading infectious disease in beef cattle production systems. Host gene expression upon facility arrival may indicate risk of BRD development and severity. However, a time-course approach would better define how BRD development influences immunological and inflammatory responses after disease occurrences. Here, we evaluated whole blood transcriptomes of high-risk beef cattle at three time points to elucidate BRD-associated host response. Sequenced jugular whole blood mRNA from 36 cattle (2015: n = 9; 2017: n = 27) across three time points (n = 100 samples; days [D]0, D28, and D63) were processed through ARS-UCD1.2 reference-guided assembly (HISAT2/Stringtie2). Samples were categorized into BRD-severity cohorts (Healthy, n = 14; Treated 1, n = 11; Treated 2+, n = 11) via frequency of antimicrobial clinical treatment. Assessment of gene expression patterns over time within each BRD cohort was modeled through an autoregressive hidden Markov model (EBSeq-HMM; posterior probability ≥ 0.5, FDR < 0.01). Mixed-effects negative binomial models (glmmSeq; FDR < 0.05) and edgeR (FDR < 0.10) identified differentially expressed genes between and across cohorts overtime. A total of 2,580, 2,216, and 2,381 genes were dynamically expressed across time in Healthy, Treated 1, and Treated 2+ cattle, respectively. Genes involved in the production of specialized resolving mediators (SPMs) decreased at D28 and then increased by D63 across all three cohorts. Accordingly, SPM production and alternative complement were differentially expressed between Healthy and Treated 2+ at D0, but not statistically different between the three groups by D63. Magnitude, but not directionality, of gene expression related to SPM production, alternative complement, and innate immune response signified Healthy and Treated 2+ cattle. Differences in gene expression at D63 across the three groups were related to oxygen binding and carrier activity, natural killer cell-mediated cytotoxicity, cathelicidin production, and neutrophil degranulation, possibly indicating prolonged airway pathology and inflammation weeks after clinical treatment for BRD. These findings indicate genomic mechanisms indicative of BRD development and severity over time.