Use of overlapping DNA pools to discern genetic differences despite pooling error.

Genotyping pools of commercial cattle and individual seedstock animals may reveal hidden relationships between sectors enabling use of commercial data for genetic evaluation. However, commercial data capture may be compromised by inexact pool formation. We aimed to estimate the concordance between distances or genomic covariance among pooling allele frequencies (PAFs) of DNA pools comprised of 100 animals with 0% or 50% overlap of animals in common between pools. Cattle lung samples were collected from a commercial beef processing plant on a single day. Six pools of 100 animals each were constructed so that overlap between pools was 0% or 50%. Two pools of all 200 animals were constructed to estimate PAFs for all 200 animals. Frozen lung tissue (0.01 g) from each animal was weighed into a tube containing a pool; there were two pools of 200 animals each and six pools of 100 animals each. Every contribution of an individual animal was an independent measurement to insure independence of pooling errors. Lung samples were kept on dried ice during the pooling process to keep them from thawing. The eight pools were then assayed for approximately 100,000 single nucleotide polymorphisms (SNP). PAF for each SNP and pool was based on the relative intensity of the two dyes used to detect the alleles rather than genotype calls which are not tractable from pooling data. Euclidean distances and genomic relationships among the PAFs for the eight pools were estimated and distances were tested for concordance with pool overlap using permutation-based analysis of distance. Distances among pools were concordant with the planned overlap of animals shared between pools (P = 0.0024); pool overlap accounted for 70% of the variation and pooling error accounted for 30%. Pools containing 100 animals with no overlap were the most distant from one another and pools with 50% overlap were the least distant. This work shows that we can discern differences in distance between pairs of overlapping DNA pools sharing 0% and 50% of the animals. Genomic correlations among nonoverlapping pools indicated that nonoverlapping pool pairs did not share many related animals because genomic correlations were near zero for these pairs. On the other hand, one pair of nonoverlapping pools likely contained related animals between pools because the correlation was 0.21. Pools sharing 50% overlap ranged in genomic relationship between 0.21 and 0.39 (N = 12).

Genetic evaluation of seedstock cattle could benefit from commercial data. There are hidden relationships between commercial and seedstock sectors because many commercial producers buy bulls from the seedstock sector. Relationships are hidden because pedigree is not tracked in commercial populations. Single nucleotide polymorphism genotypes could reveal these hidden relationships; however, genotyping can be cost prohibitive. Cost of commercial data capture could be decreased by pooling DNA which is a method to genotype groups of animals to use their data in genetic evaluation; however, error from inexact pool formation can complicate interpretation. Results from pools of overlapping random unrelated animals mimic the results from pools sharing relatives with the same degree of shared genomes. For example, a pool of progeny and a pool of the dams of the pooled progeny would produce the same result as two pools sharing 50% overlap of random unrelated animals. We can estimate the relatedness between unknown pools even in the presence of pooling error if an unknown pool comparison is similar to an overlapping pool comparison. Knowing the relationship between seedstock cattle and pools of commercial cattle may allow commercial data to enhance genetic evaluation of seedstock animals.

Genome Sequence of an Ungulate Tetraparvovirus 1 from a Nasal Swab of an Irish Beef-Suckler Calf.

Here, we report the genome sequence of strain UTPV1/AB belonging to the species Ungulate tetraparvovirus 1 (UTPV1). UTPV1/AB was isolated in the east of Ireland, directly from a nasal swab of a beef-suckler calf diagnosed with bovine respiratory disease on a farm in County Meath (longitude, 6°65'W; latitude, 53°52'N).

Polymorphism of the follicle stimulating hormone receptor does not impact reproductive performance or in-vitro embryo production in beef heifers.

Assisted reproductive technologies are used to propagate desirable genetics in a shortened timeframe. Selected females undergo ovarian stimulation with the use of follicle stimulating hormone (FSH) to increase embryo recovery for subsequent transfer programs. The FSH receptor (FSHR) c.337 C > G variant was reported to have a reduction in viable embryo numbers in an ovarian stimulation protocol. We, therefore, hypothesized that FSHR c.337 C > G would result in reduced in-vitro blastocyst development. Beef heifers were genotyped and selected based on the c.337 C > G FSHR genotype (CC, CG, GG; n = 15-16/genotype). Estrus was synchronized with a Select Synch protocol and heifers were slaughtered 5 days after induced ovulation. Anterior pituitaries, serum and reproductive tracts were collected at slaughter for analysis. Cumulus oocyte complexes (COCs) were collected and pooled within genotype for in-vitro fertilization (IVF) and subsequent blastocyst development. No differences were observed in carcass weights, anterior pituitary weights, serum progesterone, corpus lutea weight, surface follicle counts, histological follicle counts or follicular fluid estradiol concentration (P > 0.1) due to FSHR genotype. Differences were observed for ovulation rates in the GG FSHR genotype group (P < 0.01). However, cleavage and blastocyst rates were not affected due to FSHR genotype (P > 0.1), following standard IVF protocols. The FSHR variant does not influence antral follicle counts, estradiol production, or in-vitro blastocyst development in beef heifers. The GG FSHR genotype had an increased ovulation rate, which may indicate a greater potential for twinning, but research with a larger population is warranted to support this hypothesis.

Probiotics in milk replacer affect the microbiome of the lung in neonatal dairy calves.

Introduction: Probiotics have been investigated for their many health benefits and impact on the microbiota of the gut. Recent data have also supported a gut-lung axis regarding the bacterial populations (microbiomes) of the two locations; however, little research has been performed to determine the effects of oral probiotics on the microbiome of the bovine respiratory tract. We hypothesized that probiotic treatment would result in changes in the lung microbiome as measured in lung lavage fluid. Our overall goal was to characterize bacterial populations in the lungs of calves fed probiotics in milk replacer and dry rations from birth to weaning. Methods: A group of 20 dairy calves was split into two treatment groups: probiotic (TRT; N = 10, milk replacer +5 g/d probiotics; Bovamine Dairy, Chr. Hansen, Inc., Milwaukee, WI) and control (CON; N = 10, milk replacer only). On day 0, birth weight was obtained, and calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group and then included in their dry ration. Lung lavages were performed on day 52 on five random calves selected from each treatment group. DNA was extracted from lavage fluid, and 16S ribosomal RNA (rRNA) gene hypervariable regions 1-3 were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for the identification of the bacterial taxa present. Taxa were classified into both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs). Results: Overall, the evaluation of these samples revealed that the bacterial genera identified in the lung lavage samples of probiotic-fed calves as compared to the control calves were significantly different based on the OTU dataset (p < 0.05) and approached significance for the ASV dataset (p < 0.06). Additionally, when comparing the diversity of taxa in lung lavage samples to nasal and tonsil samples, taxa diversity of lung samples was significantly lower (p < 0.05). Discussion: In conclusion, analysis of the respiratory microbiome in lung lavage samples after probiotic treatment provides insight into the distribution of bacterial populations in response to oral probiotics and demonstrates that oral probiotics affect more than the gut microbiome.

Recent Emergence of Bovine Coronavirus Variants with Mutations in the Hemagglutinin-Esterase Receptor Binding Domain in U.S. Cattle.

Bovine coronavirus (BCoV) has spilled over to many species, including humans, where the host range variant coronavirus OC43 is endemic. The balance of the opposing activities of the surface spike (S) and hemagglutinin-esterase (HE) glycoproteins controls BCoV avidity, which is critical for interspecies transmission and host adaptation. Here, 78 genomes were sequenced directly from clinical samples collected between 2013 and 2022 from cattle in 12 states, primarily in the Midwestern U.S. Relatively little genetic diversity was observed, with genomes having >98% nucleotide identity. Eleven isolates collected between 2020 and 2022 from four states (Nebraska, Colorado, California, and Wisconsin) contained a 12 nucleotide insertion in the receptor-binding domain (RBD) of the HE gene similar to one recently reported in China, and a single genome from Nebraska collected in 2020 contained a novel 12 nucleotide deletion in the HE gene RBD. Isogenic HE proteins containing either the insertion or deletion in the HE RBD maintained esterase activity and could bind bovine submaxillary mucin, a substrate enriched in the receptor 9-O-acetylated-sialic acid, despite modeling that predicted structural changes in the HE R3 loop critical for receptor binding. The emergence of BCoV with structural variants in the RBD raises the possibility of further interspecies transmission.

Genome Sequence of Bovine Coronavirus Variants from the Nasal Virome of Irish Beef Suckler and Pre-weaned Dairy Calves Clinically Diagnosed with Bovine Respiratory Disease.

We report 24 bovine coronavirus (BCoV) genome sequences from Ireland. BCoV was sequenced directly from nasal swabs that had been collected during a bovine respiratory disease (BRD) outbreak among recently purchased beef suckler and pre-weaned dairy calves.

Association between clinical respiratory signs, lung lesions detected by thoracic ultrasonography and growth performance in pre-weaned dairy calves.

BACKGROUND: Bovine respiratory disease (BRD) is the main cause of mortality among 1-to-5 month old calves in Ireland, accounting for approximately one-third of deaths. Despite widespread use of clinical respiratory signs for diagnosing BRD, lung lesions are detected, using thoracic ultrasonography (TUS) or following post-mortem, in calves showing no clinical signs. This highlights the limitation of clinical respiratory signs as a method of detecting sub-clinical BRD. Using 53 purchased artificially-reared male dairy calves, the objectives of this study were to: (i) characterise the BRD incidence detected by clinical respiratory signs and/or TUS, (ii) investigate the association between clinical respiratory signs and lung lesions detected by TUS, and (iii) assess the effect of BRD on pre-weaning growth. RESULTS: Clinical BRD (based on Wisconsin clinical respiratory score and/or rectal temperature > 39.6 ºC) was detected in 43 % and sonographic changes (lung lesions) were detected in 64 % of calves from purchase (23 (SD; 6.2) days of age) until weaning, 53 days post-arrival. Calves with clinical BRD were treated. Sixty-one per cent calves affected with clinical BRD had lung lesions 10.5 days (median) before detection of clinical signs. Moderate correlations (rsp 0.70; P < 0.05) were found between cough and severe lung lesions on arrival day, and between rectal temperature > 39.6 ºC and lung lesions ≥ 2 cm2 on day 7 (rsp 0.40; P < 0.05) post-arrival. Mean average daily live weight gain (ADG) of calves from purchase to weaning was 0.75 (SD; 0.10) kg; calves with or without clinical BRD did not differ in ADG (P > 0.05), whereas ADG of those with severe lung lesions (lung lobe completely consolidated or pulmonary emphysema) was 0.12 kg/d less (P < 0.05) than calves without lung lesions. CONCLUSIONS: Thoracic ultrasonography detected lung consolidation in calves that did not show signs of respiratory disease. The presence of severe lung lesions was associated with reduced pre-weaning growth. These findings emphasise the importance of using TUS in addition to clinical respiratory scoring of calves for an early and accurate detection of clinical and sub-clinical BRD.

Evaluating Accuracy of DNA Pool Construction Based on White Blood Cell Counts.

Pooling individual samples prior to DNA extraction can mitigate the cost of DNA extraction and genotyping; however, these methods need to accurately generate equal representation of individuals within pools. The objective of this study was to determine accuracy of pool construction of blood samples based on white blood cell counts compared to two common DNA quantification methods. Fifty individual bovine blood samples were collected, and then pooled with all individuals represented in each pool. Pools were constructed with the target of equal representation of each individual animal based on number of white blood cells, spectrophotometric readings, spectrofluorometric readings, and whole blood volume with 9 pools per method and a total of 36 pools. Pools and individual samples that comprised the pools were genotyped using a commercially available genotyping array. ASReml was used to estimate variance components for individual animal contribution to pools. The correlation between animal contributions between two pools was estimated using bivariate analysis with starting values set to the result of a univariate analysis. Adonis test on distance matrix from the animal correlation showed clustering with method, and higher correlations between methods than within (P < 1 × 10-6). White blood cell count was predictive of sample representation when compared to pooling based on DNA concentration. Therefore, constructing pools using white blood cell counts prior to DNA extraction may reduce cost associated with DNA extraction and genotyping and improve representation of individuals in a pool.

Growth performance and hematological changes of weaned beef calves diagnosed with respiratory disease using respiratory scoring and thoracic ultrasonography.

This study investigated 1) the effect of clinical bovine respiratory disease (BRD) and associated lung consolidations on growth performance and hematological profiles of recently weaned beef calves and 2) the relationship between clinical respiratory signs and lung consolidation detected by thoracic ultrasonography (TUS). One hundred and fifty-three weaned beef calves (209 days old [SD: 35.8] and 306 kg [SD: 26.3], at arrival) purchased and transported from auction markets were accommodated indoors in concrete slatted floor pens. Calves were weighed weekly from arrival until day 28 and on day 65 post-arrival. Assessment of BRD and blood sample collection for hematological profiles were performed on scheduled days (at arrival, on days 7, 14, and 28) and on other days upon BRD diagnosis. Animals were assessed for BRD using a total clinical respiratory score (CRS) of five clinical signs (rectal temperature, ear position, cough, nasal secretion, and eye secretion with each ranging from normal [0] to abnormal [3]) and TUS scores (normal [0] to lung consolidation ≥ 1 cm2 [2]). Based on CRS, 35% of calves were CRS+ (CRS ≥ 5) and 65% were CRS- (CRS < 5). Although no lung consolidations (TUS-) were detected at arrival, 34% of calves developed lung consolidation (≥1 cm2) (TUS+) during the first 28 d post-arrival. Only fever (>39.6 °C) and nasal discharge were weakly associated (r = 0.19, P <0.05) with lung consolidation. On the day of BRD detection, neutrophil number and neutrophil:lymphocyte ratio were 58% and 73% greater, respectively, in BRD calves with lung consolidation compared with healthy calves. From day 0 to 65, calf average daily gain (ADG) did not differ (P >0.05) between CRS+ and CRS- calves but was 0.09 kg/d lower (P < 0.05) for TUS+ compared with TUS- calves. Calves classified as BRD (CRS + TUS ≥ 5) with lung consolidation had lower (P < 0.05) ADG from arrival until day 28 than healthy calves and BRD calves without lung consolidation (0.11 ± 0.10 vs. 0.53 ± 0.07 vs. 0.57 ± 0.10 kg/d, respectively); however, no differences in ADG were observed from day 0 to 65. Conventional methods to diagnose BRD failed to detect calves with lung lesions. TUS is a useful tool to detect lung lesions and its implementation in combination with CRS should provide a more accurate and early diagnosis of BRD, which is fundamental to successful treatment, animal welfare, and growth performance.

Classification of 16S rRNA reads is improved using a niche-specific database constructed by near-full length sequencing.

Surveys of microbial populations in environmental niches of interest often utilize sequence variation in the gene encoding the ribosomal small subunit (the 16S rRNA gene). Generally, these surveys target the 16S genes using semi-degenerate primers to amplify portions of a subset of bacterial species, sequence the amplicons in bulk, and assign to putative taxonomic categories by comparison to databases purporting to connect specific sequences in the main variable regions of the gene to specific organisms. Due to sequence length constraints of the most popular bulk sequencing platforms, the primers selected amplify one to three of the nine variable regions, and taxonomic assignment is based on relatively short stretches of sequence (150-500 bases). We demonstrate that taxonomic assignment is improved through reduced unassigned reads by including a survey of near-full-length sequences specific to the target environment, using a niche of interest represented by the upper respiratory tract (URT) of cattle. We created a custom Bovine URT database from these longer sequences for assignment of shorter, less expensive reads in comparisons of the upper respiratory tract among individual animals. This process improves the ability to detect changes in the microbial populations of a given environment, and the accuracy of defining the content of that environment at increasingly higher taxonomic resolution.

Epigenomics and genotype-phenotype association analyses reveal conserved genetic architecture of complex traits in cattle and human.

BACKGROUND: Lack of comprehensive functional annotations across a wide range of tissues and cell types severely hinders the biological interpretations of phenotypic variation, adaptive evolution, and domestication in livestock. Here we used a combination of comparative epigenomics, genome-wide association study (GWAS), and selection signature analysis, to shed light on potential adaptive evolution in cattle. RESULTS: We cross-mapped 8 histone marks of 1300 samples from human to cattle, covering 178 unique tissues/cell types. By uniformly analyzing 723 RNA-seq and 40 whole genome bisulfite sequencing (WGBS) datasets in cattle, we validated that cross-mapped histone marks captured tissue-specific expression and methylation, reflecting tissue-relevant biology. Through integrating cross-mapped tissue-specific histone marks with large-scale GWAS and selection signature results, we for the first time detected relevant tissues and cell types for 45 economically important traits and artificial selection in cattle. For instance, immune tissues are significantly associated with health and reproduction traits, multiple tissues for milk production and body conformation traits (reflecting their highly polygenic architecture), and thyroid for the different selection between beef and dairy cattle. Similarly, we detected relevant tissues for 58 complex traits and diseases in humans and observed that immune and fertility traits in humans significantly correlated with those in cattle in terms of relevant tissues, which facilitated the identification of causal genes for such traits. For instance, PIK3CG, a gene highly specifically expressed in mononuclear cells, was significantly associated with both age-at-menopause in human and daughter-still-birth in cattle. ICAM, a T cell-specific gene, was significantly associated with both allergic diseases in human and metritis in cattle. CONCLUSION: Collectively, our results highlighted that comparative epigenomics in conjunction with GWAS and selection signature analyses could provide biological insights into the phenotypic variation and adaptive evolution. Cattle may serve as a model for human complex traits, by providing additional information beyond laboratory model organisms, particularly when more novel phenotypes become available in the near future.

De novo assembly of the cattle reference genome with single-molecule sequencing.

BACKGROUND: Major advances in selection progress for cattle have been made following the introduction of genomic tools over the past 10-12 years. These tools depend upon the Bos taurus reference genome (UMD3.1.1), which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies. RESULTS: We present the new reference genome for cattle, ARS-UCD1.2, based on the same animal as the original to facilitate transfer and interpretation of results obtained from the earlier version, but applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly includes 2.7 Gb and is >250× more continuous than the original assembly, with contig N50 >25 Mb and L50 of 32. We also greatly expanded supporting RNA-based data for annotation that identifies 30,396 total genes (21,039 protein coding). The new reference assembly is accessible in annotated form for public use. CONCLUSIONS: We demonstrate that improved continuity of assembled sequence warrants the adoption of ARS-UCD1.2 as the new cattle reference genome and that increased assembly accuracy will benefit future research on this species.

Differentiation of Mannheimia haemolytica genotype 1 and 2 strains by visible phenotypic characteristics on solid media.

Genotype 2 Mannheimia haemolytica associate with the lungs of cattle with bovine respiratory disease more frequently than genotype 1 strains. Different colony colors and morphologies were identified between genotype 1 and 2 solid media cultures. Genotype of strains, and frequency differences between them in mixed cultures are discernable by visual inspection.

Microbiome of the upper nasal cavity of beef calves prior to weaning12.

Disease incidence is intimately associated with an animal's commensal bacteria populations (microbiome), as microbes that are involved with morbidity and mortality are commonly found in animals with no sign of disease. An understanding of the animal's resident respiratory pathogens, in the upper nasal cavity prior to weaning, may help us to understand the impact of these pathogens on incidence of respiratory disease. For this research, the overall goal was to characterize bacterial populations associated with calves at an early age and through time periods prior to weaning in 3 herds at the U.S. Meat Animal Research Center. Nasal swabs from the upper nasal cavity were collected at initial vaccination (approximately 40 d of age), preconditioning (approximately 130 d of age), and weaning (approximately 150 d of age) in 2015 and 2016. DNA was extracted from nasal swabs and combined into 2 pools of 10 animals for each sampling time point, in each herd, for a total of 6 pools at each sampling time point and 18 pools for all sampling time points within each year. To evaluate and compare the microbiome of each pooled sample, hypervariable regions 1 through 3 along the 16S ribosomal RNA (rRNA) gene were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for identification of the bacterial taxa present. Alpha and beta diversity were also measured. Overall, microbial communities were different between combinations of sampling year, herd location, and sampling time prior to weaning as shown by beta diversity. Analysis of these specific respiratory pathogens prior to weaning will present a clearer picture of the distribution of microbial populations in animals prior to weaning and not exhibiting clinical signs of respiratory disease. Therefore, evaluation of the animal's resident bacterial populations in the upper nasal cavity during different phases of the beef production system may help us to understand the impact of the microbiome on incidence of respiratory disease in cattle.

Longitudinal study of humoral immunity to bovine coronavirus, virus shedding, and treatment for bovine respiratory disease in pre-weaned beef calves.

BACKGROUND: Bovine coronavirus (BCV) is associated with respiratory infections in cattle of all ages; however, a temporal study to evaluate the effect of BCV immunity on virus shedding and bovine respiratory disease (BRD) incidence in pre-weaned beef calves has not been reported. Thus, we report here a prospective study in three herds of crossbred beef calves (n = 817) with endemic BCV. Serial blood samples for measurement of serum anti-BCV antibody titers and nasal swabs for detection of BCV and other common viral and bacterial BRD pathogens were collected from all calves or subsets of calves at predetermined times from birth through weaning. The calves were monitored for BRD and those that developed signs of respiratory disease were sampled for diagnostic testing. To discover additional risk factors that could have influenced BRD development, sequence analysis of the BCV strain(s) circulating in each herd, and the prevalence of common opportunistic bacterial pathogens in the upper respiratory tract of sick and apparently healthy cattle were also evaluated. RESULTS: Two hundred forty-eight of the 817 study calves (30.4%) were treated for BRD prior to weaning; 246 of those were from a single herd involved in two outbreaks of BRD leading to mass treatment of all calves in that group. Molecular diagnostic testing found BCV and Histophilus somni in nasal swabs taken at the time of BRD treatment. Between herd analyses revealed anti-BCV serum antibody abundance did not associate with the incidence of BRD or BCV shedding, though these measurements may have been hindered by the long periods between sample collections. Analysis of the BCV spike gene hypervariable region revealed four polymorphisms in 15 isolates from the three herds, making strain variation unlikely to account for differences in treatment rates between herds. Persistent or recurrent shedding episodes of BCV occurred in some animals treated for BRD. CONCLUSION: Co-detection of BCV and H. somni at the time of the disease outbreak suggests that these pathogens contributed to disease pathogenesis. Developing appropriate control measures for respiratory BCV infections may help decrease the incidence of pre-weaning BRD. The role of antibodies in protection must still be further defined.

Complete blood count data and leukocyte expression of cytokine genes and cytokine receptor genes associated with bovine respiratory disease in calves.

OBJECTIVE: The purpose of this study was to evaluate potential relationships between cytokine gene expression, complete blood counts (CBC) and animals that were sick or would become sick. The CBC and the transcript abundance of cytokines and their receptors expressed in leukocytes were measured from calves at two early timepoints, and again after diagnosis with bovine respiratory disease (BRD). RESULTS: Blood was collected from calves at pre-conditioning (n = 796) and weaning (n = 791) for CBC. Blood counts were also measured for the calves with BRD (n = 13), and asymptomatic calves (n = 75) after weaning. The CBC were compared for these animals at 3 time points. At diagnosis, neutrophils were higher and basophils lower in sick animals (P < 0.05). To further characterize BRD responses, transcript abundance of 84 cytokine genes were evaluated in 5 calves with BRD and 9 asymptomatic animals at all time points. There was more data for CBC than transcript abundance; hence, animal and temporary environmental correlations between CBC and transcript abundance were exploited to improve the power of the transcript abundance data. Expression of CCL16, CXCR1, CCR1 was increased in BRD positive animals compared to controls (P-corrected < 0.1). Cytokine expression data may help to provide insight into an animal's health.

Evaluating the microbiome of two sampling locations in the nasal cavity of cattle with bovine respiratory disease complex (BRDC).

Bovine respiratory disease complex (BRDC) is a multifactor disease, and disease incidence may be associated with an animal's commensal bacterial populations (microbiome) in the upper nasal cavity. Identifying these commensal bacterial populations in the upper nasal cavity may help us to understand the impact of the microbiome on incidence of BRDC in cattle. Various sampling techniques have previously been utilized to evaluate the microbiome of different locations of the upper nasal cavity in cattle. Therefore, our objective was to determine whether bacterial populations of the nasal cavity vary based on these sampling locations. Two common sampling techniques were evaluated, including 6-inch nasal swabs and deep nasopharyngeal swabs. Nasal swabs from calves were collected when the animal was diagnosed with BRDC after weaning in the feedlot in addition to collection of samples from asymptomatic cohorts. Samples were pooled in groups based on year the animal was in the feedlot (2015 or 2016), when the animal was diagnosed with BRDC (1 to 5 weeks after weaning), type of sample (6-inch nasal swab or deep nasopharyngeal swab), and health status (diagnosis with BRDC or control). Variable regions 1 through 3 along the 16S rRNA gene were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for identification of the bacterial taxa present. Overall, sampling site did not consistently influence diversity of the bacterial populations of the upper nasal cavity. However, the effect of disease incidence on the microbiome was depended on sampling time after weaning (P = 0.0462) for 2015, while the main effects of sampling time after weaning (P = 0.00992) and disease phenotype (P = 0.012) were significant for 2016. These data for 2016 demonstrate that in addition to bacterial profiles changing throughout weaning, calves diagnosed with BRDC have different bacterial profiles compared to their control cohorts. In addition, evaluation of the microbiome identified predominant bacteria genera in the upper nasal cavity included those previously reported to be associated with cattle diagnosed with BRDC including Mycoplasma sp., Psychrobacter sp., and Mannheimia sp. In summary, these results demonstrate that shorter, less invasive 6-inch nasal swabs produce similar results to deep nasopharyngeal swabs.

Association of Circulating Transfer RNA fragments with antibody response to Mycoplasma bovis in beef cattle.

BACKGROUND: High throughput sequencing allows identification of small non-coding RNAs. Transfer RNA Fragments are a class of small non-coding RNAs, and have been identified as being involved in inhibition of gene expression. Given their role, it is possible they may be involved in mediating the infection-induced defense response in the host. Therefore, the objective of this study was to identify 5' transfer RNA fragments (tRF5s) associated with a serum antibody response to M. bovis in beef cattle. RESULTS: The tRF5s encoding alanine, glutamic acid, glycine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with antibody response against M. bovis. tRF5s encoding alanine, glutamine, glutamic acid, glycine, histidine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with season, which could be attributed to calf growth. There were interactions (P < 0.05) between antibody response to M. bovis and season for tRF5 encoding selenocysteine (anticodon UGA), proline (anticodon CGG), and glutamine (anticodon TTG). Selenocysteine is a rarely used amino acid that is incorporated into proteins by the opal stop codon (UGA), and its function is not well understood. CONCLUSIONS: Differential expression of tRF5s was identified between ELISA-positive and negative animals. Production of tRF5s may be associated with a host defense mechanism triggered by bacterial infection, or it may provide some advantage to a pathogen during infection of a host. Further studies are needed to establish if tRF5s could be used as a diagnostic marker of chronic exposure.

Evaluation of the effect of serum antibody abundance against bovine coronavirus on bovine coronavirus shedding and risk of respiratory tract disease in beef calves from birth through the first five weeks in a feedlot.

OBJECTIVE To evaluate the effect of serum antibody abundance against bovine coronavirus (BCV) on BCV shedding and risk of bovine respiratory disease (BRD) in beef calves from birth through the first 5 weeks in a feedlot. ANIMALS 890 natural-service crossbred beef calves from 4 research herds. PROCEDURES Serial blood samples for measurement of serum anti-BCV antibody abundance by an ELISA and nasal swab specimens for detection of BCV and other viral and bacterial BRD pathogens by real-time PCR methods were collected from all calves or subsets of calves at predetermined times from birth through the first 5 weeks after feedlot entry. Test results were compared among herds, over time, and between calves that did and did not develop BRD. The associations of various herd and calf factors with test results were also evaluated. RESULTS At the calf level, serum anti-BCV antibody abundance was not associated with BCV shedding, but BCV shedding was positively associated with BRD incidence before and after weaning. The mean serum anti-BCV antibody abundance at weaning for a group of calves was inversely related with the subsequent incidence of BRD in that group; however, the serum anti-BCV antibody abundance at weaning for individual calves was not predictive of which calves would develop BRD after feedlot entry. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that serum anti-BCV antibody abundance as determined with ELISA were not associated with BCV shedding or risk of BRD in individual beef calves from birth through the first 5 weeks after feedlot entry.

Association of MicroRNAs with Antibody Response to Mycoplasma bovis in Beef Cattle.

The objective of this study was to identify microRNAs associated with a serum antibody response to Mycoplasma bovis in beef cattle. Serum from sixteen beef calves was collected at three points: in summer after calves were born, in fall at weaning, and in the following spring. All sera collected in the summer were ELISA-negative for anti-M. bovis. By the fall, eight animals were seropositive for IgG (positive group), while eight remained negative (negative group). By spring, all animals in both groups were seropositive. MicroRNAs were extracted from sera and sequenced on the Illumina HiSeq next-generation sequencer. A total of 1,374,697 sequences mapped to microRNAs in the bovine genome. Of these, 82% of the sequences corresponded to 27 microRNAs, each represented by a minimum of 10,000 sequences. There was a statistically significant interaction between ELISA response and season for bta-miR-24-3p (P = 0.0268). All sera collected at the initial summer had a similar number of copies of this microRNA (P = 0.773). In the fall, the positive group had an increased number of copies when compared to the negative group (P = 0.021), and this grew more significant by the following spring (P = 0.0001). There were 21 microRNAs associated (P< 0.05) with season. These microRNAs could be evaluated further as candidates to potentially improve productivity in cattle. The microRNAs bta-let-7b, bta-miR- 24-3p, bta-miR- 92a, and bta-miR-423-5p, were significatly associated with ELISA status (P< 0.05). These microRNAs have been recognized as playing a role in the host defense against bacteria in humans, mice, and dairy cattle. Further studies are needed to establish if these microRNAs could be used as diagnostic marker or indicator of exposure, or whether intervention strategies could be developed as an alternative to antibiotics for controlling disease due to M. bovis.