Antimicrobial resistance (AMR) is a threat to global public health and the identification of genetic determinants of AMR is a critical component to epidemiological investigations. High-throughput sequencing (HTS) provides opportunities for investigation of AMR across all microbial genomes in a sample (i.e. the metagenome). Previously, we presented MEGARes, a hand-curated AMR database and annotation structure developed to facilitate the analysis of AMR within metagenomic samples (i.e. the resistome). Along with MEGARes, we released AmrPlusPlus, a bioinformatics pipeline that interfaces with MEGARes to identify and quantify AMR gene accessions contained within a metagenomic sequence dataset. Here, we present MEGARes 2.0 (https://megares.meglab.org), which incorporates previously published resistance sequences for antimicrobial drugs, while also expanding to include published sequences for metal and biocide resistance determinants. In MEGARes 2.0, the nodes of the acyclic hierarchical ontology include four antimicrobial compound types, 57 classes, 220 mechanisms of resistance, and 1,345 gene groups that classify the 7,868 accessions. In addition, we present an updated version of AmrPlusPlus (AMR ++ version 2.0), which improves accuracy of classifications, as well as expanding scalability and usability.

Throughout the hypothalamus there are several regions known to contain sex differences in specific cellular, neurochemical, or cell grouping characteristics. The current study examined the potential origin of sex differences in calbindin expression in the preoptic area and hypothalamus as related to sources of nitric oxide. Specific cell populations were defined by immunoreactive (ir) calbindin and neuronal nitric oxide synthase (nNOS) in the preoptic area/anterior hypothalamus (POA/AH), anteroventral periventricular nucleus (AVPv), and ventromedial nucleus of the hypothalamus (VMN). The POA/AH of adult mice was characterized by a striking sex difference in the distribution of cells with ir-calbindin. Examination of the POA/AH of androgen receptor deficient Tfm mice suggests that this pattern was in part androgen receptor dependent, since Tfm males had reduced ir-calbindin compared with wild-type males and more similar to wild-type females. At P0 ir-calbindin was more prevalent than in adulthood, with males having significantly more ir-calbindin and nNOS than have females. Cells that contained either ir-calbindin or ir-nNOS in the POA/AH were in adjacent cell groups, suggesting that NO derived from the enzymatic activity of nNOS may influence the development of ir-calbindin cells. In the region of AVPv, at P0, there was a sex difference with males having more ir-nNOS fibers than have females while ir-calbindin was not detected. In the VMN, at P0, ir-nNOS was greater in females than in males, with no significant difference in ir-calbindin. We suggest that NO as an effector molecule and calbindin as a molecular biomarker illuminate key aspects of sexual differentiation in the developing mouse brain.

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The preoptic area/anterior hypothalamus (POA/AH) is characterized by sexually dimorphic features in a number of vertebrates and is a key region of the forebrain for regulating physiological responses and sexual behaviors. Using live-cell, fluorescent video microscopy with organotypic brain slices the current study examined sex differences in the movement characteristics of neurons expressing yellow fluorescent protein (YFP) driven by the Thy-1 promoter. Cells in slices from embryonic day 14 (E14), but not E13 mice displayed significant sex differences in their basal neuronal movement characteristics. Exposure to 10nM estradiol-17β (E2), but not 100nM dihydrotestosterone, significantly altered cell movement characteristics within minutes of exposure in a location specific manner. E2 treatment decreased the rate of motion of cells located in the dorsal POA/AH, but increased the frequency of movement in cells located more ventrally. These effects were consistent across age and sex. To further determine whether early developing sex differences in the POA/AH depend upon gonadal steroids, we examined cell positions in mice with a disruption of the steroidogenic factor-1 gene in which gonads do not form. An early born cohort of cells was labeled with the mitotic indicator bromodeoxyuridine (BrdU) on E11. More cells were found in the POA/ AH of females than males on the day of birth (P0) regardless of gonadal status. These results support the hypothesis that estrogen partially contributes to brain sexual dimorphism through its influence on cell movements during development. Estrogen's influence may be superimposed upon a preexisting genetic bias.

Ruminants are a critical human food source and have been implicated as a potentially important source of global methane emissions. Because of their unique digestive physiology, ruminants rely upon a symbiotic relationship with the complex and rich community of microorganism in the foregut to allow digestion of complex carbohydrates. This study used 16S rRNA gene sequencing to investigate the composition of microbial communities from three rumen micro-environments of cattle fed identical diets: (1) free fluid, (2) the fibrous pack, and (3) the mucosa. Community composition analysis revealed that while a phylogenetic core including the most abundant and most common ruminal taxa (members of Bacteroidetes and Firmicutes) existed across micro-environments, the abundances of these taxa differed significantly between fluid- and mucosa-associated communities, and specific lineages were discriminant of individual micro-environments. Members of Firmicutes, specifically Clostridiales, Lachnospiraceae, Mogibacteriaceae, Christenellaceae, and Erysipelotrichaceae were significantly more abundant in fluid communities, while members of Bacteroidetes, namely Muribaculaceae and Prevotellaceae were more abundant in mucosa-associated communities. Additionally, Methanobacteriaceae, a family of methanogenic Archaea, was more abundant in fluid-associated communities. A set of four more diverse lineages were discriminant of pack-associated communities that included Succinivibrionaceae, RFP12 (Verruco-5), Fibrobacteraceae, and Spirochaetaceae. Our findings indicate that different ecological niches within each micro-environment have resulted in significant differences in the diversity and community structure of microbial communities from rumen fluid, pack, and mucosa without the influence of diet that will help contextualize the influence of other environmental factors.

Background Bovine respiratory disease (BRD) is caused by interactions among host, environment, and pathogens. One standard method for antemortem pathogen identification in cattle with BRD is deep-guarded nasopharyngeal swabbing, which is challenging, costly, and waste generating. The objective was to compare the ability to recover Mannheimia haemolytica and compare microbial community structure using 29.5 inch (74.9 cm) deep-guarded nasopharyngeal swabs, 16 inch (40.6 cm) unguarded proctology swabs, or 6 inch (15.2 cm) unguarded nasal swabs when characterized using culture, real time-qPCR, and 16S rRNA gene sequencing. Samples for aerobic culture, qPCR, and 16S rRNA gene sequencing were collected from the upper respiratory tract of cattle 2 weeks after feedlot arrival. Results There was high concordance of culture and qPCR results for all swab types (results for 77% and 81% of sampled animals completely across all 3 swab types for culture and qPCR respectively). Microbial communities were highly similar among samples collected with different swab types, and differences identified relative to treatment for BRD were also similar. Positive qPCR results for M. haemolytica were highly concordant (81% agreed completely), but samples collected by deep-guarded swabbing had lower amounts of Mh DNA identified (Kruskal–Wallis analysis of variance on ranks, P < 0.05; Dunn-test for pairwise comparison with Benjamini–Hochberg correction, P < 0.05) and lower frequency of positive compared to nasal and proctology swabs (McNemar’s Chi-square test, P < 0.05). Conclusions Though differences existed among different types of swabs collected from individual cattle, nasal swabs and proctology swabs offer comparable results to deep-guarded nasopharyngeal swabs when identifying and characterizing M. haemolytica by culture, 16S rRNA gene sequencing, and qPCR.

IntroductionThe objectives of this study were to evaluate the impacts of two modified-live virus (MLV) vaccination protocols and respiratory disease (BRD) occurrence on the microbial community composition of the nasopharynx in feedlot cattle.MethodsThe treatment groups included in this randomized controlled trial included: 1) no viral respiratory vaccination (CON), 2) intranasal, trivalent, MLV respiratory vaccine in addition to a parenteral BVDV type I and II vaccine (INT), and 3) parenteral, pentavalent, MLV respiratory vaccination against the same agents (INJ). Calves (n = 525) arrived in 5 truckload blocks and were stratified by body weight, sex, and presence of a pre-existing identification ear-tag. A total of 600 nasal swab samples were selected for DNA extraction and subsequent 16S rRNA gene sequencing to characterize the microbiome of the upper respiratory tract. Nasal swabs collected on d 28 from healthy cattle were used to evaluate the impact of vaccination on upper respiratory tract (URT) microbial communities.ResultsFirmicutes were less abundant in INT calves (n = 114; P &lt; 0.05) and this difference was attributed to decreased relative abundance (RA) of Mycoplasma spp. (P = 0.04). Mannheimia and Pasteurella had lower RA in INT (P &lt; 0.05). The microbiome in healthy animals on d 28 had increased Proteobacteria (largely Moraxella spp.) and decreased Firmicutes (comprised almost exclusively of Mycoplasma spp.) compared to animals that were treated for or died from BRD (P &lt; 0.05). Cattle that died had a greater RA of Mycoplasma spp. in their respiratory microbiome on d 0 (P &lt; 0.02). Richness was similar on d 0 and 28, but diversity increased for all animals on d 28 (P&gt;0.05).

Background The potential to distribute bacteria resistant to antimicrobial drugs in the meat supply is a public health concern. Market cows make up a fifth of the U.S. beef produced but little is known about the entire population of bacteria (the microbiome) and entirety of all resistance genes (the resistome) that are found in this population. The objective of this study was to characterize and compare the resistomes and microbiome of beef, dairy, and organic dairy market cows at slaughter. Methods Fifty-four (N = 54) composite samples of both colon content and meat trimmings rinsate samples were collected over six visits to two harvest facilities from cows raised in three different production systems: conventional beef, conventional dairy, and organic dairy (n = 3 samples per visit per production system). Metagenomic DNA obtained from samples were analyzed using target-enriched sequencing (resistome) and 16S rRNA gene sequencing (microbiome). Results All colon content samples had at least one identifiable antimicrobial resistance gene (ARG), while 21 of the 54 meat trimmings samples harbored at least one identifiable ARGs. Tetracycline ARGs were the most abundant class in both colon content and carcass meat trimmings. The resistome found on carcass meat trimmings was not significantly different by production system (P = 0.84, R2 = 0.00) or harvest facility (P = 0.10, R2 = 0.09). However, the resistome of colon content differed (P = 0.01; R2 = 0.05) among production systems, but not among the harvest facilities (P = 0.41; R2 = 0.00). Amplicon sequencing revealed differences (P < 0.05) in microbial populations in both meat trimmings and colon content between harvest facilities but not production systems (P > 0.05). Conclusions These data provide a baseline characterization of an important segment of the beef industry and highlight the effect that the production system where cattle are raised and the harvest facilities where an animal is processed can impact associated microbiome and resistomes.